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PubMed2006

Allelic variants of ABC drug transporter Cdr1p in clinical isolates of Candida albicans.

Candida drug resistance protein (Cdr1p) is a major drug efflux protein, which plays a key role in commonly encountered clinical azole resistance in Candida albicans. We have analyzed its sequence in several azole resistant clinical isolates to evaluate the allelic variation within CDR1 gene and to relate it to its functional activity. The sequence analysis revealed 53 single nucleotide polymorphisms (SNPs), out of which six were non-synonymous single nucleotide polymorphisms (NS-SNPs) implying a change in amino acid and were found in two or more than two allelic combinations in different sensitive or resistant isolates. We have identified three new NS-SNPs namely, E948P, T950S, and F1399Y, in isolates wherein F1399Y appeared to be unique and was present in one of the naturally occurring azole resistant isolates obtained from Indian diabetic patients. However, site-directed mutagenesis showed that the residue F1399 in between TMS 11 and TMS 12 does not affect the functionality of Cdr1p. Taken together, our SNPs analyses reveal that unlike human P-gp, the naturally acquired allelic variations are mostly present in non-conserved regions of the protein which do not allow Cdr1p to genetically evolve in a manner, that would allow a change in its functionality to affect substrate recognition, specificity, and drug efflux activity of C. albicans cells.

Alleles↗
PubMed2003

Structural location of disease-associated single-nucleotide polymorphisms.

Non-synonymous single-nucleotide polymorphism (nsSNP) of genes introduces amino acid changes to proteins, and plays an important role in providing genetic functional diversity. To understand the structural characteristics of disease-associated SNPs, we have mapped a set of nsSNPs derived from the online mendelian inheritance in man (OMIM) database to the structural surfaces of encoded proteins. These nsSNPs are disease-associated or have distinctive phenotypes. As a control dataset, we mapped a set of nsSNPs derived from SNP database dbSNP to the structural surfaces of those encoded proteins. Using the alpha shape method from computational geometry, we examine the geometric locations of the structural sites of these nsSNPs. We classify each nsSNP site into one of three categories of geometric locations: those in a pocket or a void (type P); those on a convex region or a shallow depressed region (type S); and those that are buried completely in the interior (type I). We find that the majority (88%) of disease-associated nsSNPs are located in voids or pockets, and they are infrequently observed in the interior of proteins (3.2% in the data set). We find that nsSNPs mapped from dbSNP are less likely to be located in pockets or voids (68%). We further introduce a novel application of hidden Markov models (HMM) for analyzing sequence homology of SNPs on various geometric sites. For SNPs on surface pocket or void, we find that there is no strong tendency for them to occur on conserved residues. For SNPs buried in the interior, we find that disease-associated mutations are more likely to be conserved. The approach of classifying nsSNPs with alpha shape and HMM developed in this study can be integrated with additional methods to improve the accuracy of predictions of whether a given nsSNP is likely to be disease-associated.

Disease↗
PubMed2001

Identification of a novel retrovirus long terminal repeat (LTR) that is targeted by p51A (TAp63gamma) and selective dominant-negative activity of p73L (DeltaNp63alpha) toward p53-responsive promoter activities.

The p51/p73L/p63/p40 gene, recently identified as a p53 homolog, encodes two major isoforms, p51A and p73L, which are suggested to have similar functions synonymous with p53 and dominant-negative activity toward both p53 and p51A, respectively. We have cloned a high affinity genomic fragment bound to p51A that was assigned to be a novel retrovirus long terminal repeat. Strikingly, this fragment was found to bind to both p53 and p73L with similar affinity to p51A. Additional demonstration with known p53 response elements suggested that DNA-binding profiles of p51A and p73L were very similar but were distinct from that of p53. Consistent with this novel finding, transient cotransfection experiments in mammalian cells suggested that p73L, when it was expressed at a low level, selectively suppressed p53-dependent transactivation of p21-luc and mdm2-luc but not of cyclinG-luc and bax-luc reporters. These data raise the possibility that p73L differentially modulates the p53 function according to the distinct DNA-binding affinity between these two proteins.

Animals↗
PubMed2000

Covariation of GC content and the silent site substitution rate in rodents: implications for methodology and for the evolution of isochores.

Many attempts to test selectionist and neutralist models employ estimates of synonymous (Ks) and non-synonymous (Ka) substitution rates of orthologous genes. For example, a stronger Ka-Ks correlation than expected under neutrality has been argued to indicate a role for selection and the absence of a Ks-GC4 correlation has been argued to be inconsistent with neutral models for isochore evolution. However, both of these results, we have shown previously, are sensitive to the method by which Ka and Ks are estimated. Using a maximum likelihood (ML) estimator (GY94) we found a positive correlation between Ks and GC4 and only a weak correlation between Ka and Ks, lower than expected under neutral expectations. This ML method is computationally slow. Recently, a new ad hoc approximation of this ML method has been provided (YN00). This is effectively an extension of Li's protocol but that also allows for codon usage bias. This method is computationally near-instantaneous and therefore potentially of great utility for analysis of large datasets. Here we ask whether this method might have such applicability. To this end we ask whether it too recovers the two unusual results. We report that when the ML and earlier ad hoc methods disagree, YN00 recovers the results described by the ML methods, i.e. a positive correlation between GC4 and Ks and only a weak correlation between Ks and Ka. If the ML method can be trusted, then YN00 can also be considered an adequately reliable method for analysis of large datasets. Assuming this to be so we also analyze further the patterns. We show, for example, that the positive correlation between GC4 and Ks is probably in part a mutational bias, there being more methyl induced CpG-->TpG mutations in GC rich regions. As regards the evolution of isochores, it seems inappropriate to use the claimed lack of a correlation between GC and Ks as definitive evidence either against or for any model. If the positive correlation is real then, we argue, this is hard to reconcile with the biased gene conversion model for isochore formation as this predicts a negative correlation.

Animals↗
PubMed2005

Genomic characterization of KIR2DL4 in families and unrelated individuals reveals extensive diversity in exon and intron sequences including a common frameshift variation occurring in several alleles.

The KIR2DL4 gene including a portion of exon 1 through exon 9 was sequenced from two families and eight cell lines from the International Histocompatibility Workshop (IHWS). Two known alleles and eight variants were detected. Overall, there were five synonymous and three non-synonymous changes when the variants were compared to the coding sequences of the most closely related known alleles plus a common frameshift change in five of the variant alleles. Alignment of the new variants with all known alleles showed that the regions encoding the extracellular region and the cytoplasmic tail were the most polymorphic. Two non-synonymous changes, P146H and L161V, occurred in an extracellular immunoglobulin-like domain. Five of the eight variants had a single adenine deletion in the exon encoding the transmembrane region, potentially resulting in a truncated protein lacking the cytoplasmic tail. The distribution of the deletion variant among many KIR2DL4 alleles may explain the high frequency of this variation in the population. Four of the eight consanguineous IHWS cell lines were found to be heterozygous for KIR2DL4 carrying two alleles that differed from one another by a few nucleotide substitutions. Analysis of intron sequences in the families revealed the nature and distribution of interspersed repeat elements which comprise 46% of the KIR2DL4 nucleotide sequence and consist of 12 elements including six SINEs (13.73% of the total length), one LINE (12.41%), and five LTR elements (19.51%). The results revealed the presence of extensive diversity in the KIR2DL4 gene. This is the first extensive report providing both exon and intron data in related individuals.

Alleles↗
PubMed1989

NTP Toxicology and Carcinogenesis Studies of Tribromomethane (Bromoform) (CAS No. 75-25-2) in F344/N Rats and B6C3F1 Mice (Gavage Studies).

Tribromomethane, a chemical intermediate and solvent, has been identified as a drinking water contaminant resulting from water chlorination. Toxicology and carcinogenesis studies were conducted by administering tribromomethane (95%-97% pure) in corn oil by gavage to groups of F344/N rats and B6C3F1 mice of each sex once or for 14 days, 13 weeks, or 2 years. Single-Administration, Fourteen-Day, and Thirteen-Week Studies: All rats that received 2,000 mg/kg and 3/5 males and 3/5 females that received 1,000 mg/kg tribromomethane died before the end of the single-administration studies. All mice that received 2,000 mg/kg, 4/5 males and 2/5 females that received 1,000 mg/kg, and 1/5 males that received 500 mg/kg died before the end of the studies. Shallow breathing was observed for rats and male mice that received 1,000 or 2,000 mg/kg tribromomethane. In the 14-day studies, all rats that received 600 or 800 mg/kg and 1/5 males that received 400 mg/kg tribromomethane died before the end of the studies. The final mean body weight of male rats that received 400 mg/kg was 14% lower than that of vehicle controls. One of five male mice that received 600 mg/kg and 1/5 female mice that received 800 mg/kg died before the end of the studies. Final mean body weights of dosed and vehicle control mice were comparable. None of the rats died before the end of the 13-week studies (doses ranged from 12 to 200 mg/kg). Final mean body weights were comparable for dosed and vehicle control rats. All male rats that received 100 or 200 mg/kg tribromomethane and all female rats that received 200 mg/kg were lethargic. The incidences of cytoplasmic vacuolization of hepatocytes in dosed male rats were slightly increased compared with that in vehicle controls. The severity of this lesion was increased in the 200 mg/kg group. One of 10 female mice that received 100 mg/kg tribromomethane died before the end of the 13-week studies. The final mean body weight of mice that received 400 mg/kg was 8% lower than that of vehicle controls for males and was comparable to that of vehicle controls for females. Cytoplasmic vacuolization of hepatocytes was observed in the liver of 5/10 male mice that received 200 mg/kg and in 8/10 male mice that received 400 mg/kg tribromomethane. Based on these results, 2-year studies of tribromomethane were conducted by administering 0, 100, or 200 mg/kg tribromomethane in corn oil by gavage, 5 days per week for 103 weeks, to groups of 50 F344/N rats of each sex and 50 female B6C3F1 mice. Male B6C3F1 mice were administered 0, 50, or 100 mg/kg tribromomethane on the same schedule. Body Weights and Survival in the Two-Year Studies: Mean body weights of high dose male and female rats were 10%-28% lower than those of vehicle controls throughout the second year of the studies. Survival of the high dose group of male rats was significantly lower than that of the vehicle controls after week 91; no significant differences in survival were observed between any groups of female rats (male: vehicle control, 34/50; low dose, 30/50; high dose, 11/50; female: 34/50; 28/50; 28/50). Reduced survival for male rats given 200 mg/kg tribromomethane lowered the sensitivity of this group to detect a carcinogenic response. Mean body weights of dosed and vehicle control male mice were comparable throughout the study. Mean body weights of dosed female mice were 5%-16% lower than those of vehicle controls from week 28 to the end of the study. No significant differences in survival were observed between any groups of male mice; the survival of both dosed groups of female mice was significantly lower than that of the vehicle controls after week 77 (male: 41/50; 37/50; 36/50; female: 25/49; 15/50; 20/50). Reduced survival in all groups of female mice was partly due to a utero-ovarian infection; nonetheless, survival of all groups of female mice was at least 50% by week 92. Nonneoplastic and Neoplastic Effects in the Two-Year Studies: Uncommon adenomatous polyps or adenocarcinomas (combined) of the large intestine (colon or rectum) were induced in three male ratarge intestine (colon or rectum) were induced in three male rats (vehicle control, 0/50; low dose, 0/50; high dose, 3/50) and in nine female rats (0/50; 1/50; 8/50); the historical incidence of neoplasms of the large intestine is less than 0.2% in approximately 2,000 corn oil vehicle control male F344/N rats, and none has been observed in approximately 2,000 corn oil vehicle control female F344/N rats. Three of the neoplasms of the large intestine (one in the high dose male rats and two in the high dose female rats) were adenocarcinomas. Focal or diffuse fatty change of the liver was observed at increased incidences in dosed rats (male: 23/50; 49/50; 50/50; female: 19/50; 39/49; 46/50). Active chronic inflammation was observed at increased incidences in dosed male and high dose female rats (male: 0/50; 29/50; 23/50; female: 9/50; 8/49; 27/50). The incidence of necrosis of the liver was increased in high dose male rats (7/50; 3/50; 20/50) and decreased in dosed females (11/50; 3/49; 2/50). Mixed cell focus was observed at increased incidences in dosed female rats (8/50; 25/49; 28/50). Other nonneoplastic lesions observed at increased incidences in dosed rats included chronic active inflammation and squamous metaplasia of the ducts of the salivary gland (squamous metaplasia-- male: 0/50; 15/50; 31/48; female: 0/49; 10/49; 16/50; chronic active inflammation--male: 0/50; 16/50; 25/48; female: 0/49; 9/49; 18/50), squamous metaplasia of the prostate gland (2/49; 6/46; 12/50), ulcers of the forestomach (male: 1/49; 5/50; 10/50), and chronic active inflammation of the lung (male: 1/50; 7/50; 15/50). Pigmentation of the spleen was also observed at an increased incidence in high dose female rats. The salivary gland and lung lesions were characteristic of infection by rat coronavirus, a virus to which a positive serologic reaction was observed early in the studies. The incidence of follicular cell hyperplasia of the thyroid gland was increased in high dose female mice (5/49; 4/49; 19/47), and fatty change of the liver was increased in both dosed groups of female mice (1/49; 9/50; 24/50). No chemically related adverse effects were observed in male mice. Neoplastic lesions that occurred at lower incidences in dosed animals compared with those in vehicle controls included preputial gland neoplasms in male rats (10/41; 5/38; 1/34), uterine stromal polyps in female rats (10/49; 9/50; 2/50), anterior pituitary gland adenomas in male and female rats (male: 12/50; 12/48; 2/45; female: 29/48; 12/46; 16/48), mammary gland fibroadenomas in female rats (22/50; 17/50; 6/50), and alveolar/bronchiolar neoplasms in male mice (11/50; 7/50; 2/49). Other than concomitant decreases in body weights, no other reasons are obvious to correlate these decreases with chemical administration. Genetic Toxicology: Tribromomethane exhibited equivocal mutagenicity in Salmonella typhimurium strain TA100 in the absence of exogenous metabolic activation and in strains TA97 and TA98 when exposure occurred in the presence of hamster S9; tribromomethane produced no increases in revertant colonies in TA1535 or TA1537 with or without exogenous metabolic activation. Tribromomethane induced trifluorothymidine (Tft) resistance in mouse L5178Y cells with and without metabolic activation. When tested incultured Chinese hamster ovary (CHO) cells for cytogenetic effects, tribromomethane produced an increase in both sister chromatid exchanges (SCEs) and chromosomal aberrations in the absence, but not in the presence, of exogenous metabolic activation. Tribromomethane caused sex-linked recessive lethal mutations in Drosophila when administered to adult males by feeding; no induction of mutations was observed when tribromomethane was administered by abdominal injection. Results of tests for reciprocal translocations in adult male Drosophila exposed to tribromomethane by feeding were negative. In vivo tests for cytogenetic effects in bone marrow cells of male B6C3F1 mice demonstrated that intraperitoneal injection of tribromomethane induced an increase in SCEs but no increase in chromosomal aberrations. Intraperitoneal injection of tribromomethane also induced an increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of B6C3F1 mice. Audit: The data, documents, and pathology materials from the 2-year studies of tribromomethane have been audited. The audit findings show that the conduct of the studies is documented adequately and support the data and results given in this Technical Report. Conclusions: Under the conditions of these 2-year gavage studies, there was some evidence of carcinogenic activity of tribromomethane for male F344/N rats and clear evidence of carcinogenic activity for female F344/N rats, based on increased incidences of uncommon neoplasms of the large intestine. Reduced survival for male rats given 200 mg/kg tribromomethane lowered the sensitivity of this group to detect a carcinogenic response. Chemically related nonneoplastic lesions included fatty change and active chronic inflammation of the liver in male and female rats, minimal necrosis of the liver in male rats, and mixed cell foci of the liver in female rats. There was no evidence of carcinogenic activity for male B6C3F1 mice given 50 or 100 mg/kg tribromomethane or for female B6C3F1 mice given 100 or 200 mg/kg; male mice might have been able to tolerate a higher dose. Survival of female mice was reduced, partly due to a utero-ovarian infection. Synonym: bromoform

Journal Article↗
PubMed1995

Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes.

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (KN) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The KN values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (KA) were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

Amino Acid Sequence↗
PubMed2004

Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans.

Phytophthora infestans, the organism responsible for the Irish famine, causes late blight, a re-emerging disease of potato and tomato. Little is known about the molecular evolution of P. infestans genes. To identify candidate effector genes (virulence or avirulence genes) that may have co-evolved with the host, we mined expressed sequence tag (EST) data from infection stages of P. infestans for secreted and potentially polymorphic genes. This led to the identification of scr74, a gene that encodes a predicted 74-amino acid secreted cysteine-rich protein with similarity to the Phytophthora cactorum phytotoxin PcF. The expression of scr74 was upregulated approximately 60-fold 2 to 4 days after inoculation of tomato and was also significantly induced during early stages of colonization of potato. The scr74 gene was found to belong to a highly polymorphic gene family within P. infestans with 21 different sequences identified. Using the approximate and maximum likelihood (ML) methods, we found that diversifying selection likely caused the extensive polymorphism observed within the scr74 gene family. Pairwise comparisons of 17 scr74 sequences revealed elevated ratios of nonsynonymous to synonymous nucleotide-substitution rates, particularly in the mature region of the proteins. Using ML, all 21 polymorphic amino acid sites were identified to be under diversifying selection. Of these 21 amino acids, 19 are located in the mature protein region, suggesting that selection may have acted on the functional portions of the proteins. Further investigation of gene copy number and organization revealed that the scr74 gene family comprises at least three copies located in a region of no more than 300 kb of the P. infestans genome. We found evidence that recombination contributed to sequence divergence within at least one gene locus. These results led us to propose an evolutionary model that involves gene duplication and recombination, followed by functional divergence of scr74 genes. This study provides support for using diversifying selection as a criterion for identifying candidate effector genes from sequence databases.

Algal Proteins↗
PubMed1999

NTP Toxicology and Carcinogenesis Studies of Ethylbenzene (CAS No. 100-41-4) in F344/N Rats and B6C3F1 Mice (Inhalation Studies).

Ethylbenzene is mainly used in the manufacture of styrene. Ethylbenzene is also a major component of mixed xylenes used as solvents in agricultural and home insecticide sprays, rubber and chemical manufacturing, and household degreasers, paints, adhesives, and rust preventives. Ethylbenzene is also used as an antiknock agent in aviation and motor fuels. Ethylbenzene was nominated for study by the National Institute for Occupational Safety and Health (NIOSH) and the Occupational Safety and Health Administration (OSHA) because of its potential for widespread human exposure and because of its structural similarity to benzene and toluene. Male and female F344/N rats and B6C3F1 mice were exposed to ethylbenzene (greater than 99% pure) by inhalation for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, mouse lymphoma cells, cultured Chinese hamster ovary cells, and mouse peripheral blood erythrocytes. In previously reported 13-week toxicity studies in which F344/N rats and B6C3F1 mice were exposed to ethylbenzene by whole body inhalation exposure, no histopathologic changes were observed (NTP, 1992). 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were exposed to 0, 75, 250, or 750 ppm ethylbenzene by inhalation, 6 hours per day, 5 days per week, for 104 weeks. Survival, Body Weights, and Clinical Findings Survival of male rats in the 750 ppm group was significantly less than that of the chamber controls. Mean body weights of 250 and 750 ppm males were generally less than those of the chamber controls beginning at week 20. Mean body weights of exposed groups of females were generally less than those of chamber controls during the second year of the study. Pathology Findings In male rats exposed to 750 ppm, the incidences of renal tubule adenoma and adenoma or carcinoma (combined) were significantly greater than the chamber control incidences. In addition, the incidence of renal tubule hyperplasia in 750 ppm males was significantly greater than that in the chamber controls. The findings from an extended evaluation (step section) of the kidneys showed a significant increase in the incidences of renal tubule adenoma and hyperplasia in 750 ppm males and females; the incidence of renal tubule adenoma or carcinoma (combined) was significantly increased in 750 ppm males. The severities of nephropathy in 750 ppm male and all exposed female rats were significantly increased relative to the chamber controls. The incidence of interstitial cell adenoma in the testis of 750 ppm males was significantly greater than that in the chamber control group and slightly exceeded the historical control range for inhalation studies. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were exposed to 0, 75, 250, or 750 ppm ethylbenzene by inhalation, 6 hours per day, 5 days per week, for 103 weeks. Survival, Body Weights, and Clinical Findings Survival of exposed groups of male and female mice was similar to that of the chamber controls. Mean body weights of female mice exposed to 75 ppm were greater than those of the chamber controls from week 72 until the end of the study. Pathology Findings In 750 ppm males, the incidences of alveolar/ bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly greater than those in the chamber control group but were within the NTP historical control ranges. The incidence of alveolar epithelial metaplasia in 750 ppm males was significantly greater than that in the chamber controls. In 750 ppm females, the incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) were significantly greater than those in the chamber control group but were within the historical control ranges. The incidence of eosinophilic foci in 750 ppm females was significantly increased compared to that in the chamber controls. There was a spectrum of nonneoplastic liver changes related to ethylbenzene exposure in male mice, including syncytial alteration of hepatocytes, hepatocellular hypertrophy, and hepatocyte necrosis. rosis. The incidences of hyperplasia of the pituitary gland pars distalis in 250 and 750 ppm females and the incidences of thyroid gland follicular cell hyperplasia in 750 ppm males and females were significantly increased compared to those in the chamber control groups. GENETIC TOXICOLOGY: Ethylbenzene gave little indication of mutagenicity, in vitro or in vivo. No induction of mutations was noted in Salmonella typhimurium strain TA97, TA98, TA100, or TA1535 with or without S9 metabolic activation, and no increases in sister chromatid exchanges or chromosomal aberrations were observed in cultured Chinese hamster ovary cells treated with ethylbenzene, with or without S9. In the mouse lymphoma assay, a significant mutagenic response was noted in the absence of S9, but only at the highest nonlethal dose tested and with accompanying cytotoxicity; the test was not performed with S9. No increases in the frequency of micronucleated erythrocytes were observed in vivo in peripheral blood samples from male and female mice exposed to ethylbenzene for 13 weeks. CONCLUSIONS: Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity of ethylbenzene in male F344/N rats based on increased incidences of renal tubule neoplasms. The incidences of testicular adenoma were also increased. There was some evidence of carcinogenic activity of ethylbenzene in female F344/N rats based on increased incidences of renal tubule adenomas. There was some evidence of carcinogenic activity of ethylbenzene in male B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. There was some evidence of carcinogenic activity of ethylbenzene in female B6C3F1 mice based on increased incidences of hepatocellular neoplasms. Exposure of male and female rats to ethylbenzene resulted in increased incidences of renal tubule hyperplasia and increased severities of nephropathy. Exposure of male mice to ethylbenzene resulted in increased incidences of alveolar epithelial metaplasia, syncytial alteration of hepatocytes, hepatocellular hypertrophy, hepatocyte necrosis, and thyroid gland follicular cell hyperplasia. In female mice, ethylbenzene exposure resulted in increased incidences of eosinophilic foci of the liver, pituitary gland pars distalis hyperplasia, and thyroid gland follicular cell hyperplasia. Synonyms: EB; ethylbenzol; phenylethane

Journal Article↗
PubMedDate not supplied

Mu opioid receptor gene variants: lack of association with alcohol dependence.

The mu opioid receptor is implicated in the reward, tolerance and withdrawal effects of alcohol and other drugs of abuse. This hypothesis is supported by the effects of alcohol on beta-endorphin release, of mu opioid receptor agonists and antagonists on alcohol consumption, and by the activation of the dopaminergic reward system by both alcohol and opiates. In addition, the murine mu opioid receptor locus, Oprm, is implicated as the major quantitative trait locus (QTL) affecting the different levels of morphine consumption between two inbred mouse strains that also exhibit differences in alcohol and cocaine consumption. Detection of genetic variation affecting OPRM1 expression or mu opioid receptor function would be an important step towards understanding the origins of inter-individual variation in response to mu opioid receptor ligands and in diseases of substance dependence. We directly sequenced the human mu opioid receptor locus, OPRM1, to detect natural variation that might affect function and/or be associated with psychiatric phenotypes related to opioid function. Four DNA sequence variants were found: three non-synonymous substitutions (Ala6Val [rare], Asn40Asp, [0.10-0.16], Ser147Cys [rare]) and one intronic variant (IVS2+691G/C [0.55-0.63]). OPRM1 alleles, genotypes and haplotypes from three psychiatrically characterized population samples (US Caucasian [USC, n=100], Finnish Caucasian [FC, n=324] and Southwestern American Indian [SAI, n=367]), were used to perform association and sib-pair linkage analyses with alcohol and drug dependence diagnoses. No significant association of OPRM1 genetic variation to phenotype was observed. This analysis has 80% power to detect a small to moderate effect of OPRM1 variation on alcohol dependence and 100% power to detect effects of the magnitude of the ALDH2*2 variant. While these data do not support a role of the mu opioid receptor in susceptibility to alcohol dependence, the potential relationship between OPRM1 genetic variation and response to endogenous opioids and exogenous opiates can now be investigated.

Adult↗
PubMed2000

Sequence variations of the hypervariable region of hepatitis C virus and their clinical significance.

OBJECTIVE: To understand the clinical significance of sequence variations in the hypervariable region (HVR) of hepatitis C virus during infection. METHODS: Eight patients with acute hepatitis C and 20 patients with chronic hepatitis C were followed up for two years. Blood samples were taken at intervals of six months for analysis of HCV-HVR sequences by reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing methods. RESULTS: HCV-HVR sequences of the 28 patients changed in various degrees. 92% of these nucleotide substitutions led to changes of corresponding aminoacid sequences. Only 8% of changed nucleotide were synonymous substitutions. Of 27 amino acids variation of amino acid ranged from 1 to 20 (mean 8, 30%). The most common nucleotide substitution (62%) occurred in the first position of codon, 31% in the second and the rest in the third. HVR variation rate was 0.89 x 10(-1) per genome site per year in acute hepatitis C, compared with 2.31 x 10(-1) per genome site per year in chronic hepatitis C (P < 0.05), but had no relation to HCV subtype. Variation of HVR in the flare up type (ALT > 150 mu/L) was much more than that in the quiescent type (ALT < 100 mu/L). CONCLUSION: Our results suggested that sequence variation of HVR during HCV chronic infection seems to be an adaptive response to HCV to evade the host immune pressure and might play a major role in the establishment of persistent infection as well as in the flare-up of hepatitis.

Acute Disease↗
PubMed2004

Microevolution and history of the plague bacillus, Yersinia pestis.

The association of historical plague pandemics with Yersinia pestis remains controversial, partly because the evolutionary history of this largely monomorphic bacterium was unknown. The microevolution of Y. pestis was therefore investigated by three different multilocus molecular methods, targeting genomewide synonymous SNPs, variation in number of tandem repeats, and insertion of IS100 insertion elements. Eight populations were recognized by the three methods, and we propose an evolutionary tree for these populations, rooted on Yersinia pseudotuberculosis. The tree invokes microevolution over millennia, during which enzootic pestoides isolates evolved. This initial phase was followed by a binary split 6,500 years ago, which led to populations that are more frequently associated with human disease. These populations do not correspond directly to classical biovars that are based on phenotypic properties. Thus, we recommend that henceforth groupings should be based on molecular signatures. The age of Y. pestis inferred here is compatible with the dates of historical pandemic plague. However, it is premature to infer an association between any modern molecular grouping and a particular pandemic wave that occurred before the 20th century.

Animals↗
PubMed1993

Toxicology and Carcinogenesis Studies of Mercuric Chloride (CAS No. 7487-94-7) in F344 Rats and B6C3F1 Mice (Gavage Studies).

Mercuric chloride is an inorganic compound that has been used in agriculture as a fungicide, in medicine as a topical antiseptic and disinfectant, and in chemistry as an intermediate in the production of other mercury compounds. The widespread use of mercury has resulted in increased levels of mercury in rivers and lakes. Mercuric chloride was evaluated in toxicity and carcinogenicity studies because of its extensive use and its occurrence as an environmental pollutant, and because of the lack of adequate long-term rodent studies. Toxicology and carcinogenesis studies were conducted by administering mercuric chloride (greater than 99% pure) in deionized water by gavage to groups of F344 rats or B6C3F1 mice for 16 days, 6 months, and 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium (strains TA98, TA100, TA1535, and TA1537), in mouse lymphoma L5178Y cells, in Chinese hamster ovary cells, and in Drosophila melanogaster. 16-DAY STUDIES: Groups of five rats of each sex received 0, 1.25, 2.5, 5, 10, or 20 mg mercuric chloride/kg body weight and groups of five mice of each sex received 0, 5, 10, 20, 40, or 80 mg/kg in deionized water by gavage for 12 dose days. Two male rats in the 20 mg/kg group died in the first week, as did all male and four female mice from the 80 mg/kg group and one male mouse from the 40 mg/kg group. The final mean body weight of male rats receiving 20 mg/kg was 10% lower than that of the controls; the final mean body weight of 20 mg/kg females was 9% lower than that of the controls. Final mean body weights and body weight gains of dosed mice were similar to those of the controls. Absolute and relative kidney weights of male rats receiving 2.5 mg/kg or greater doses and of female rats administered 5 mg/kg or more were significantly greater than those of the controls. Absolute kidney weights of mice were significantly increased in all male dose groups and in the 40 mg/kg female dose group; relative kidney weights of dosed male and female mice were significantly greater than the controls. Analysis of kidney, liver, and brain tissues for mercury residues revealed that the highest mercury concentration was in the kidneys of rats and mice. Acute renal tubule nephropathy occurred in dosed rats and was slightly more severe in males than in females. Chemical-related lesions in mice included renal tubule necrosis, inflammation and necrosis of the forestomach, and necrosis of the glandular stomach. 6-MONTH STUDIES: Groups of 10 rats of each sex received 0, 0.312, 0.625, 1.25, 2.5, or 5 mg mercuric chloride/kg body weight in deionized water by gavage for 26 weeks. Groups of 10 mice of each sex received 0, 1.25, 2.5, 5, 10, or 20 mg/kg in deionized water by gavage for 26 weeks (males) or 27 weeks (females). No deaths related to mercuric chloride administration occurred in rats or mice. Mean body weight gains of male rats that received 5 mg/kg and all female rat dose groups that received 0.625 mg/kg or greater were significantly lower than the controls. The final mean body weight and body weight gain of male mice in the 20 mg/kg group were significantly lower than those of the controls; final mean body weights and body weight gains of other dosed male mice and all dosed female mice were similar to those of the controls. Absolute and relative kidney weights of all dosed male rats and of female rats that received 0.625 mg/kg or greater were significantly greater than those of the controls. In male mice, absolute kidney weights in the three highest dose groups were significantly increased; no biologically significant differences in organ weights occurred in female mice. Analysis of kidney, liver, and brain tissues for mercury residues revealed the highest mercury concentration in the kidneys of rats and mice. The severity of chronic nephropathy increased with dose in male rats. Cytoplasmic vacuolation of renal tubule epithelial cells was observed in male mice in the 5, 10, and 20 mg/kg groups. No histopathologic changes in female mice were related to chemical exposure. 2-YEAR STUDIES: Groups YEAR STUDIES: Groups of 60 rats of each sex received 0, 2.5, or 5 mg mercuric chloride/kg body weight and groups of 60 mice of each sex received 0, 5, or 10 mg/kg in deionized water by gavage 5 days per week for 2 years. The doses were based on decreased weight gains in rats receiving 10 and 20 mg/kg and the decreased weight in male mice receiving 40 mg/kg during the 16-day studies, and on the decreased weight gains and toxicity results seen in the 6-month studies. Increased absolute and relative kidney weights in rats and male mice in the 6-month studies and degenerative renal changes suggested that higher dose levels would result in inadequate survival rates for a 2-year study. 15-Month Interim Evaluations: Relative kidney weights were significantly increased in dosed rats and female mice. The severity of nephropathy was increased in male rats, but not in females. High-dose male and female rats had minimal to mild hyperplasia of the basal cell layer in the forestomach epithelium (diagnosed as acanthosis); this lesion was not found in control or low-dose rats. Male mice had an increased severity of vacuolation of the renal tubule epithelium; no chemical-related lesions occurred in the kidneys of females. The incidence of inflammation of the olfactory epithelium lining the nasal cavity increased in male and female high-dose mice. Survival, Body Weights, and Clinical Signs: Survival of dosed male rats was lower than that of the controls (26/50, 10/50, 5/50); survival of dosed female rats was similar to that of the controls (35/50, 28/49, 30/50). Although more than 60&percnt; of the mice in each dose group survived to study end, survival rates of high-dose male mice and dosed female mice were lower than those of the controls (males: 36/50, 36/50, 31/50; females: 41/50, 35/50, 31/50). The final mean body weights of high-dose male and female rats were 15&percnt; and 14&percnt; lower than controls, respectively. The mean body weight of low-dose female rats was generally similar to controls throughout the 2-year study; the mean body weight of low-dose male rats was similar to that of the control through week 89. In mice, mean body weights of all male and female dose groups were similar to those of the controls throughout the studies. Pathology Findings: Chronic nephropathy appeared to develop at an accelerated rate and led to decreased survival in both dosed male rat groups. Secondary effects of renal dysfunction in dosed male rats resulted in increased incidences of fibrous osteodystrophy of the bone, mineralization of various tissues, and parathyroid gland hyperplasia. Based on evaluations of single and step sections, the incidence of renal tubule hyperplasia was increased in high-dose male rats (control, 3/50; high-dose, 10/50), but the incidences of renal tubule adenoma in high-dose and control males were similar (4/50, 5/50). Renal tubule hyperplasia was also slightly increased in high-dose female rats (2/50, 5/50) and adenomas were seen in high-dose females, but not in controls (0/50, 2/50). Incidences of forestomach hyperplasia in rats were markedly increased in dosed males (3/49, 16/50, 35/50) and high-dose females (5/50, 5/49, 20/50). Squamous cell papillomas of the forestomach were found in 3 low-dose and 12 high-dose males and in 2 high-dose females. No squamous cell carcinomas were found. The incidence of thyroid follicular cell carcinoma was marginally increased in high-dose male rats (1/50, 2/50, 6/50). However, a corresponding increased incidence in follicular cell adenomas (1/50, 4/50, 0/50) or hyperplasia (2/50, 4/50, 2/50) in males did not occur, and the overall incidence of follicular cell neoplasms was not significantly increased (2/50, 6/50, 6/50). The incidence of nasal mucosa inflammation in male and female rats was increased in the high-dose groups (male: 9/50, 8/47, 18/50; female: 0/49, 5/49, 15/50) and may have been related to chemical administration. The incidences of mammary gland fibroadenomas were significantly decreased in dosed female rats (15/50, 5/48, 2/50). The incidence and severity of nephropathy were increased in dosed mice; secondary effects of renal dysfunction did not occur. Renal tubule hyperplasia was found in one control and two high-dose male mice. Two renal tubule adenomas and one renal tubule adenocarcinoma were seen in high-dose male mice. Additional step sections revealed focal hyperplasia in another control male; no additional renal tubule neoplasms were found in high-dose or control males. Proliferative lesions of the renal tubule epithelium were not found in female mice. The incidence of metaplasia of the olfactory epithelium increased with dose in male and female mice. No other biologically significant lesions were found. GENETIC TOXICOLOGY: Mercuric chloride was not mutagenic in Salmonella typhimurium strains TA100, TA1535, TA1537, or TA98 with or without exogenous metabolic activation (S9). Induction of sex-linked recessive lethal mutations in germ cells of male Drosophila melanogaster did not occur when mercuric chloride was administered in feed or by injection. However, positive results were obtained in mutagenicity tests with mammalian cells. In the absence of S9, mercuric chloride induced trifluorothymidine resistance in mouse L5178Y cells and chromosomal aberrations in Chinese hamster ovary cells. In the Chinese hamster ovary cell test for induction of sister chromatid exchanges, mercuric chloride produced a negative response without S9 and a weakly positive response in the presence of S9. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was some evidence of carcinogenic activity of mercuric chloride in male F344 rats based on the increased incidence of squamous cell papillomas of the forestomach. Marginally increased incidences of thyroid follicular cell adenomas and carcinomas may have been related to mercuric chloride exposure. There was equivocal evidence of carcinogenic activity of mercuric chloride in female F344 rats based on two squamous cell papillomas of the forestomach. There was equivocal evidence of carcinogenic activity of mercuric chloride in male B6C3F1 mice based on the occurrences of two renal tubule adenomas and one renal tubule adenocarcinoma. There was no evidence of carcinogenic activity of mercuric chloride in female B6C3F1 mice receiving 5 or 10 mg/kg. Nonneoplastic lesions associated with exposure to mercuric chloride included increased severity of nephropathy in male rats and male and female mice. There was an increased incidence of renal tubule hyperplasia in male rats. The incidence of forestomach hyperplasia was increased in dosed male and female rats. Increased incidences of nasal mucosa inflammation were associated with mercuric chloride administration in rats. Increased incidences of olfactory epithelial metaplasia in mice were also associated with mercuric chloride administration. Synonyms: Abavit B, calochlor, corrosive sublimate, dichloromercury, mercuric bichloride, mercury chloride, mercury (II) chloride, mercury bichloride, mercury perchloride, sublimate, sulem, bichloride of mercury, corrosive mercury chloride, perchloride of mercury, mercury dichloride Trade name: Fungchex

Journal Article↗
PubMed2005

Characterization of the 1918 influenza virus polymerase genes.

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.

Adaptation, Physiological↗
PubMed1988

NTP Toxicology and Carcinogenesis Studies of Nitrofurazone (CAS No. 59-87-0) in F344/N Rats and B6C3F1 Mice (Feed Studies).

Nitrofurazone is a synthetic furan derivative, active against a broad spectrum of bacteria, which has been widely used in veterinary and human medicine. Toxicology and carcinogenesis studies were conducted by feeding diets containing nitrofurazone (99% pure) to groups of F344/N rats and B6C3F1 mice of each sex for 14 days, 13 weeks, or 2 years. Fourteen-Day and Thirteen-Week Studies: Groups of five males and five females of each species were fed diets containing 0, 630, 1,250, 2,500, 5,000, or 10,000 ppm for 14 consecutive days. Early deaths occurred in all groups of rats receiving 5,000 or 10,000 ppm nitrofurazone. The surviving rats in the lower two dose groups gained weight, but weight gain was decreased as the dose of nitrofurazone was increased. Feed consumption by rats of each sex was decreased at all doses above 630 ppm. In all dosed groups, clinical signs of toxicity included rough hair coats and lethargy. At doses of 2,500 ppm and above, rats of each sex exhibited intermittent episodes of seizures and lethargy. All mice that received 2,500, 5,000, or 10,000 ppm nitrofurazone and 3/5 males that received 1,250 ppm died before the end of the 14-day studies; the surviving dosed mice (except females at 630 ppm) lost weight. A dose-related decrease in feed consumption was observed at all doses above 630 ppm. Clinical signs included rough hair coats and convulsive seizures. In the 13-week studies, groups of 10 rats of each sex were given diets containing 0, 150, 310, 620, 1,250, or 2,500 ppm nitrofurazone. No deaths were observed and all animals gained weight, but the magnitude of weight gain was dose dependent with decrements in final mean body weight for the highest dose group reaching 55% in males and 36% in females. Other evidence of chemically related toxicity included convulsive seizures, osteoporosis, degenerative arthropathy, and gonadal hypoplasia in both sexes at the two highest doses. Groups of 10 mice of each sex were given diets containing 0, 70, 150, 310, 620, or 1,250 ppm nitrofurazone for 13 weeks. Early deaths were observed in the two highest dose groups of each sex. The final mean body weights of male and female mice in the 1,250-ppm groups were about 20% lower than those of the controls; weight gains of the other dosed mice were comparable to those of the controls. Stimulus-induced convulsive seizures were observed for all mice in the two highest dose groups. Testicular hypoplasia was observed in the two highest dose groups of male mice. Body Weight and Survival in the Two-Year Studies: Dietary concentrations for the 2-year studies were 0, 310, or 620 ppm for rats and 0, 150, or 310 ppm for mice (50 animals per dose group). Mean body weights of high dose male rats were lower than those of the controls after week 39; mean body weights of low dose male rats and of the controls were comparable throughout the study. Final mean body weights of low and high dose female rats were 9% and 21% lower than those of the controls. Dosed rats consumed less feed than did the controls. The average amount of nitrofurazone consumed per day was approximately 11-12 or 24-26 mg/kg by low or high dose male and female rats. The survival of the high dose group of male rats was lower than that of the controls after week 92 (final survival-- male: control, 33/50; low dose, 30/50; high dose, 20/50; female: 28/50; 37/50; 31/50). Mean body weights of dosed mice were similar to or somewhat greater than those of controls throughout most of the studies. The average daily feed consumption by dosed mice was similar to that of controls. The average amount of nitrofurazone consumed per day was approximately 14-16 or 29-33 mg/kg for low or high dose male and female mice. The survival of the high dose group of male mice was lower than that of the controls after week 88 (final survival-- male: 39/50; 31/50; 27/50; female: 39/50; 40/50; 35/50). In mice of each sex, nitrofurazone administration induced stimulus-sensitive convulsive seizures beginning at week 4 or 5 for high dose mice and week 24 for low dose female mice. These seizures were low dose female mice. These seizures were observed primarily in the first year of the study. Nonneoplastic and Neoplastic Effects in the Two-Year Studies: Degenerative changes involving the vertebral and femoro-tibial (knee) joints were observed at increased incidences in dosed rats. The degenerative changes primarily affected the articular cartilage and were similar to those seen in the 13-week studies. Degeneration of the sternal synchondroses was increased in high dose female rats. The osteoporosis seen in the 13-week studies was not observed in the 2-year studies. Testicular degeneration, characterized by atrophy of the germinal epithelium and aspermatogenesis, was observed at increased incidences in dosed male rats (control, 12/50; low dose, 49/50; high dose, 47/50). Adenomas of the sebaceous glands and trichoepitheliomas or sebaceous adenomas (combined) of the skin were observed in high dose male rats (0/50; 0/50; 5/50). Carcinomas of the preputial gland were increased in dosed male rats (1/50; 8/50; 5/50). The incidences of preputial gland adenomas or carcinomas (combined) in dosed male rats were not statistically greater than that in the controls (9/50; 16/50; 7/50). However, in the low dose group, the incidence is greater than the highest incidence observed in historical untreated control groups (9/50). In addition, hyperplasia of the preputial gland was observed in six low dose male rats in which neither adenomas nor carcinomas occurred. The incidence of mesotheliomas of the tunica vaginalis in low dose male rats was greater than that in the controls (0/50; 7/50; 2/50). Fibroadenomas of the mammary gland occurred at markedly increased incidences in dosed female rats (8/49; 36/50; 36/50). Three adenocarcinomas were also observed (1/49; 0/50; 2/50). Ovarian atrophy (7/47; 44/50; 38/50) and tubular cell hyperplasia of the ovary (1/47; 23/50; 21/50) were observed at markedly increased incidences in dosed female mice. The incidences of benign mixed tumors (0/47; 17/50; 20/50), granulosa cell tumors (1/47; 4/50; 9/50), and granulosa cell tumors or luteomas (combined) (3/47; 6/50; 9/50) of the ovary were increased in exposed female mice. Mononuclear cell leukemia in rats occurred with negative trends (male: 21/50; 23/50; 6/50; female: 15/49; 2/50; 2/50). In female mice, the incidences of adenomas or carcinomas (combined) of the anterior pituitary gland occurred with a negative trend (10/50; 7/50; 2/49). The incidences of testicular interstitial cell tumors were decreased in dosed male rats (45/50; 30/50; 28/50). Genetic Toxicity: Nitrofurazone was mutagenic in Salmonella typhimurium strains TA98 and TA100 both with and without exogenous metabolic activation. The responses in strains TA1535 and TA1537 were more varied: nitrofurazone was mutagenic in strain TA1535 only in the presence of S9 and produced no consistent increase in gene reversions in strain TA1537 with or without S9. In the absence of metabolic activation, nitrofurazone induced forward mutations at the TK+/- locus of mouse L5178Y lymphoma cells; the chemical was not tested with S9. Treatment of cultured Chinese hamster ovary cells with nitrofurazone in the absence of S9 produced a dose-related increase in sister chromatid exchanges and chromosomal aberrations; with S9, sister chromatid exchanges were increased, but no induction of chromosomal aberrations was observed. Audit: The data, documents, and pathology materials from the 2-year studies of nitrofurazone were audited at the NTP Archives. The audit findings show that the conduct of the studies is documented adequately and support the data and results given in this Technical Report. Conclusions: Under the conditions of these 2-year feed studies, there was equivocal evidence of carcinogenic activity of nitrofurazone for male F344/N rats as shown by the occurrence of sebaceous gland adenomas and trichoepitheliomas of the skin, mesotheliomas of the tunica vaginalis, and preputial gland tumors. There was clear evidence of carcinogenic activity of nitrofurazone for female F344/N rats as shown by a markedly increased incidence of fibroadenomas of the mammary gland. There was no evidence of carcinogenic activity for male B6C3F1 mice fed diets containing nitrofurazone at concentrations of 150 or 310 ppm. There was clear evidence of carcinogenic activity of nitrofurazone for female B6C3F1 mice as shown by increased incidences of benign mixed tumors and granulosa cell tumors of the ovary. Administration of nitrofurazone was associated with decreased incidences of mononuclear cell leukemia in male and female rats, testicular interstitial cell tumors in male rats, and pituitary gland neoplasms in female mice. Convulsive seizures in mice of each sex, ovarian atrophy in female mice, testicular degeneration in rats, and degeneration of articular cartilage in rats were all associated with the administration of nitrofurazone. Synonyms: 5-nitro-2-furaldehyde semicarbazone; 2-[(5-nitro-2-furanyl)methylene]hydrazine carboximide Trade Names: Aldomycin; Amifur; Chemfuran; Coxistat; Furacin; Furacinetten; Furaplast; Furazol W; Furesol; Furracoccid; Mammex; Nefco; Nifuzon; Nitrofural; Vabrocid

Journal Article↗
PubMed1989

NTP Toxicology and Carcinogenesis Studies of Nitrofurantoin (CAS No. 67-20-9) in F344/N Rats and B6C3F1 Mice (Feed Studies).

Nitrofurantoin was studied and evaluated because of its widespread use as a drug for treating urinary tract infections in humans, its structural relationship to known carcinogenic 5-nitrofuran compounds, and the lack of adequate studies to assess its carcinogenicity. Toxicology and carcinogenesis studies of nitrofurantoin were conducted by administering nitrofurantoin (greater than 99% pure) in feed to groups of F344/N rats and B6C3F1 mice of each sex for 14 days, 13 weeks, or 2 years. Fourteen-Day and Thirteen-Week Studies: None of the rats (at dietary concentrations up to 20,000 ppm) died before the end of the 14-day studies. Rats that received 5,000, 10,000, or 20,000 ppm lost weight. Four of five male and 4/5 female mice that received 10,000 ppm and 1/5 females that received 5,000 ppm nitrofurantoin died before the end of the studies. Mice that received 5,000 ppm and male mice that received 10,000 ppm lost weight. In the 13-week studies, final mean body weights of rats that received 2,500, 5,000, or 10,000 ppm were 10%, 34%, or 47% lower than that of the controls for males and 15%, 31%, or 41% lower for females. Feed consumption by dosed and control rats was generally similar. Degeneration of the germinal epithelium of the seminiferous tubules of the testis was observed in male rats that received 2,500 to 10,000 ppm nitrofurantoin. Necrosis of the ovarian follicles was observed in 8/10 female rats that received 10,000 ppm, in 3/10 females that received 5,000 ppm, and in 1/10 that received 2,500 ppm. For mice, final mean body weights of the 5,000-ppm groups were 13% lower than that of the controls for males and 15% lower for females. Two of 10 male mice that received 5,000 ppm and 1/10 males that received 300 ppm died before the end of the 13-week studies. Estimated feed consumption was similar for dosed and control groups. Degeneration of the germinal epithelium of the testis was observed in males that received 1,300 to 5,000 ppm; necrosis of the ovarian follicles was observed in females that received 5,000 ppm but not in the lower dose groups. Necrosis of the renal tubular epithelium was observed in 2/9 males that received 5,000 ppm. Based on these results, 2-year studies of nitrofurantoin were conducted by feeding diets containing 0, 1,300, or 2,500 ppm nitrofurantoin to groups of 50 male F344/N rats and to groups of 50 male and female B6C3F1 mice for 103 weeks. Groups of 50 female F344/N rats were fed diets containing 0, 600, or 1,300 ppm nitrofurantoin on the same schedule. Body Weight and Survival in the Two-Year Studies: Mean body weight and average daily feed consumption of dosed male and female rats were similar to those of the controls throughout the studies. The average amount of nitrofurantoin consumed per day was estimated to be 60 and 110 mg/kg for low and high dose male rats and 30 and 60 mg/kg for low and high dose female rats. No significant differences in the number of rats surviving to the end of the studies were observed between any groups of rats of either sex (male: control, 24/50; low dose, 27/50; high dose, 26/50; female: 25/50; 26/50; 31/50). Mean body weights of high dose male and female mice were up to 12% lower than those of the controls throughout most of the studies. The average daily feed consumption by dosed mice ranged from 93% to 100% that by controls. The average amount of nitrofurantoin consumed per day was estimated to be 280-300 mg/kg and 570-580 mg/kg for low and high dose mice. The survival of the control group of female mice was lower than that of the dosed groups (control, 19/50; low dose, 37/50; high dose, 37/50). The decrease in survival was most likely related to the increase in microbial infection in the reproductive tract observed in the controls. Groups of male mice had similar survival (28/50; 29/50; 34/50). Nonneoplastic and Neoplastic Effects in the Two-Year Studies: Organs showing toxicity from nitrofurantoin exposure identified in the short-term studies were the testis in male rats and mice, the ovary in female rats and mice, and the kidney in male mice. Lesions oband mice, the ovary in female rats and mice, and the kidney in male mice. Lesions observed in the 2-year studies were in the testis in male rats and mice, ovary in female mice, and kidney in male rats. Chronic nephropathy was observed in nearly all rats, but the severity of the lesions was judged to be greater in dosed male rats. Hyperplasia of the transitional cell epithelium (control, 0/50; low dose, 5/50; high dose, 2/50) and hydronephrosis of the renal pelvis (0/50; 5/50; 2/50) were also observed in dosed male rats. In the standard single sections of the left and right kidney from each rat, tubular cell adenomas were observed in one low dose and two high dose males; a tubular cell carcinoma was observed in another high dose male. Because the number of renal tubular cell neoplasms identified by standard procedures in the dosed male rats was low, additional step-sections of the kidney were evaluated. The incidences of tubular cell adenomas derived from the step-sections and original sections (combined) were significantly increased in dosed male rats (adenomas: 3/50; 11/50; 19/50); tubular cell carcinomas occurred in two high dose males only. Lesions considered to be associated with the nephropathy and nitrofurantoin exposure were observed in male rats and included hyperplasia of the parathyroid glands (3/49; 18/47; 23/49), fibrous osteodystrophy of the bone (0/50; 5/50; 5/50), and mineralization of the glandular stomach (1/49; 8/50; 14/50). Atypical cells of the epididymis (0/50; 0/50; 12/50) and degeneration of the testis (0/50; 0/50; 36/50) were observed in high dose male rats. Fibrinoid necrosis of arterioles (1/50; 8/50; 15/50) and perivascular infiltration of mononuclear cells (3/50; 9/50; 19/50) were also observed in the testis of male rats. Interstitial cell adenomas of the testis occurred with a negative trend (47/50; 45/50; 21/50), and no adenomas or carcinomas of the preputial gland were seen in high dose male rats (12/48; 11/50; 0/47). The incidence of clitoral gland neoplasms was increased in low dose female rats (5/44; 10/38; 4/42). Osteosarcomas were observed in the bone of one low dose and two high dose male rats. The historical incidence of osteosarcomas in untreated male F344/N rats is 8/1,937 (0.4&percnt;). The incidences of subcutaneous tissue neoplasms in dosed male rats were greater than that in the controls (1/50; 7/50; 5/50). No neoplastic lesions in dosed female rats or male mice were considered to be compound related at the doses of nitrofurantoin administered. For female mice, ovarian atrophy was observed in 48/50 low dose and 49/50 high dose mice but not in controls. Tubular cell adenomas of the ovary (0/50; 0/50; 5/50), benign mixed tumors (tubular and stromal) (0/50; 0/50; 4/50), and granulosa cell tumors (0/50; 3/50; 2/50)) were observed in dosed female mice. One granulosa cell tumor in the high dose group was malignant. Ovarian abscesses (18/50) and suppurative inflammation of the uterus (11/50) were observed in control female mice but not in dosed female mice and are believed to be related to indigenous microbial infections and most likely were the cause of early deaths in this group. Adenocarcinomas of the uterus were seen in one low dose and in one high dose mouse. Testicular aspermatogenesis (1/49; 1/49; 16/50), degeneration of the germinal epithelium (0/49; 3/49; 23/50), and atypical cells (0/50; 0/49; 26/50) and depletion (1/50; 1/49; 15/50) of the epididymis were observed at increased incidences in high dose male mice. Spindle cell hyperplasia of the adrenal cortex was observed in dosed female mice (3/50; 41/50; 45/50). A spindle cell adenoma (adrenal capsule adenoma) was seen in one low dose female mouse, and a spindle cell carcinoma (adrenal capsule carcinoma) was seen in one low dose male mouse. Mineralization of the renal medulla (male: 0/50; 0/50; 17/50; female: 0/50; 0/50; 7/50) and dilatation of the renal tubules (male: 0/50; 0/50; 14/50) were observed in high dose mice. Hepatocellular neoplasms (adenomas or carcinomas, combined) were observed at an increased incidence in high dose female mice (2/50; 2/50; 8/50). An Ito cell tumor of the liver was observed in one low dose and one high dose female mouse. Malignant lymphomas occurred in female mice (12/50; 19/50; 24/50). Genetic Toxicology: Nitrofurantoin was mutagenic in Salmonella typhimurium strains TA98 and TA100, with and without metabolic activation, but was not mutagenic for strains TA1535 or TA1537. Nitrofurantoin induced forward mutations at the TK+/- locus of L5178Y mouse lymphoma cells in the absence of metabolic activation (it was not tested with activation). Nitrofurantoin induced increased numbers of sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary cells with and without metabolic activation. Results of the sex-linked recessive lethal assay in Drosophila were negative after administration of nitrofurantoin by feeding or by injection. Conclusions: Under the conditions of these 2-year feed studies, there was some evidence of carcinogenic activity of nitrofurantoin for male F344/N rats as shown by increased incidences of uncommon kidney tubular cell neoplasms. Uncommon osteosarcomas of the bone and neoplasms of the subcutaneous tissue were observed in dosed male rats. Incidences of interstitial cell adenomas of the testis and neoplasms of the preputial gland were decreased in the 2,500-ppm group of male rats. There was no evidence of carcinogenic activity of nitrofurantoin for female F344/N rats fed diets containing 600 ppm or 1,300 ppm for 2 years. Female rats may have been able to tolerate higher doses. There was no evidence of carcinogenic activity of nitrofurantoin for male B6C3F1 mice fed diets containing 1,300 ppm or 2,500 ppm for 2 years. There was clear evidence of carcinogenic activity of nitrofurantoin for female B6C3F1 mice as shown by increased incidences of tubular adenomas, benign mixed tumors, and granulosa cell tumors of the ovary. Nonneoplastic lesions considered related to nitrofurantoin exposure were chronic nephropathy and associated lesions (hyperplasia of the parathyroid gland, fibrous osteodystrophy of the bone, and mineralization of the glandular stomach) in male rats and testicular degeneration in male rats and mice. Ovarian atrophy and hyperplasia of the adrenal cortex spindle cells were observed in dosed female mice. Synonyms: 1-(((5-nitro-2-furanyl)methylene)amino-2,4-imidazolidinedione); 1-(5-nitro-2-furfurylideneamino)-hydantoin; N-(5-nitro-2-furfurlidene)-1-aminohydantoin; 1-((5-nitrofurfurylidene)amino)hydantoin Trade Names: Benkfuran; Benkfurin; Chemiofuran; Cyantin; Dantafur; Furadantin; Furadantine; Furadantoin; Furadonin; Furadonine; Furantoin; Furatoin; Furobactina; Ituran; Macrodantin; Nifurantin; NSC 2107; N-Toin; Orafuran; Parafuran; Urizept; USAF EA-2; Welfurin; Zoofurin

Journal Article↗
PubMed2006

Association of sixty-one non-synonymous polymorphisms in forty-one hypertension candidate genes with blood pressure variation and hypertension.

We previously selected a group of hypertension candidate genes by a key word search using the OMIM database of NCBI and validated 525 coding single nucleotide polymorphisms (SNPs) in 179 hypertension candidate genes by DNA sequencing in a Japanese population. In the present study, we examined the association between 61 non-synonymous SNPs and blood pressure variations and hypertension. We used DNA samples taken from 1,880 subjects in the Suita study, a population-based study using randomly selected subjects. Analyses of covariance adjusting for age, body mass index, hyperlipidemia, diabetes, smoking, drinking, and antihypertensive medication revealed that 17 polymorphisms in 16 genes (APOB, CAST, CLCNKB, CTNS, GHR, GYS1, HF1, IKBKAP, KCNJ11, LIPC, LPL, P2RY2, PON2, SLC4A1, TRH, VWF) were significantly associated with blood pressure variations. Multivariate logistic regression analysis with adjustment for the same factors revealed that 11 polymorphisms in 11 genes (CAST, CTLA4, F5, GC, GHR, LIPC, PLA2G7, SLC4A1, SLCI8A1, TRH, VWF) showed significant associations with hypertension. Five polymorphisms in five genes, CAST(calpastatin), LIPC (hepatic lipase), SLC4A1 (band 3 anion transporter), TRH (thyrotropin-releasing hormone), and VWF (von Willebrand factor), were significantly associated with both blood pressure variation and hypertension. Thus, our study suggests that these five genes were susceptibility genes for essential hypertension in this Japanese population.

Aged↗
PubMed1993

NTP technical report on the toxicity studies of Ethylene Glycol Ethers: 2-Methoxyethanol, 2-Ethoxyethanol, 2-Butoxyethanol (CAS Nos. 109-86-4, 110-80-5, 111-76-2) Administered in Drinking Water to F344/N Rats and B6C3F1 Mice.

Glycol alkyl ethers represent a class of high-production-volume chemicals with widespread industrial applications as solvents and chemical intermediates. Comparative toxicity studies with three glycol ethers, 2-methoxyethanol, 2-ethoxyethanol, and 2-butoxyethanol, were conducted in F344/N rats and B6C3F1 mice in both 2-week and 13-week drinking water studies. Toxicologic endpoints evaluated in animals included histopathology, hematology, clinical chemistry, urinalysis, and reproductive system parameters. Genetic toxicity was also evaluated for each glycol ether in several in vitro and in vivo assays. In the 2-week studies, groups of five male and five female rats and mice received 2-methoxyethanol, 2-ethoxyethanol, or 2-butoxyethanol in the drinking water. Estimates of compound consumption based on water consumption by male and female rats ranged from 100 to 400 mg/kg for 2-methoxyethanol, 200 to 1600 mg/kg for 2-ethoxyethanol, and 70 to 300 mg/kg for 2-butoxyethanol. For mice, consumption values ranged from 200 to 1300 mg/kg for 2-methoxyethanol, 400 to 2800 mg/kg for 2-ethoxyethanol, and 90 to 1400 mg/kg for 2-butoxyethanol. There were no chemical-related effects on survival for rats or mice in the 2-week studies. Decreased body weight gains were noted for both male and female rats treated with 2-methoxyethanol or 2-ethoxyethanol for 2 weeks, and there were dose-related decreases in water consumption for rats of each sex treated with the ethylene glycol ethers. Most of the changes in organ weights for rats and mice treated with the glycol ethers were sporadic (mice) or related to low final mean body weights (rats), except for thymic atrophy in male and female rats and testicular atrophy in males of both species receiving 2-methoxyethanol or 2-ethoxyethanol. In the 13-week studies in rats, groups of 10 males and 10 females received 2-methoxyethanol, 2-ethoxyethanol, or 2-butoxyethanol in the drinking water at concentrations ranging from 750 to 6000 ppm, 1250 to 20,000 ppm, or 750 to 6000 ppm, respectively. In the 13-week studies in mice, groups of 10 males and 10 females received 2-methoxyethanol, 2-ethoxyethanol, or 2-butoxyethanol in the drinking water at concentrations ranging from 2000 to 10,000 ppm, 2500 to 40,000 ppm, or 750 to 6000 ppm, respectively. Estimates of compound consumption based on water consumption by male and female rats ranged from 70 to 800 mg/kg for 2-methoxyethanol, 100 to 2200- mg/kg for 2-ethoxyethanol, and 70 to 500 mg/kg for 2-butoxyethanol. For-mice, consumption values ranged from 300 to 1800 mg/kg for 2-methoxyethanol, 600 to 11,000 mg/kg for 2-ethoxyethanol, and 100 to 1300 mg/kg for 2-butoxyethanol. Chemical-related mortality occurred in male and female rats administered 4500 or 6000 ppm 2-methoxyethanol and in male and female rats administered 20,000 ppm 2-ethoxyethanol. No deaths occurred in rats administered 2-butoxyethanol or in mice administered 2-methoxyethanol, 2-ethoxyethanol, or 2-butoxyethanol. Decreased body weight gains occurred in dosed rats and mice in all three studies; the greatest reductions in body weight gain were seen with 2-methoxyethanol. In rats administered 2-methoxyethanol or 2-ethoxyethanol, treatment-related histopathologic changes were observed in the testes, thymus, and hematopoietic tissues (spleen, bone marrow, and liver). A dose-related degeneration of the germinal epithelium in the seminiferous tubules of the testes was more severe in 2-methoxyethanol-treated rats than in rats treated with 2-ethoxyethanol. In special stop-exposure studies in male rats in which administration of the glycol ethers was stopped after 60 days, marked degeneration of the seminiferous tubules was present in rats treated with 3000 ppm 2-methoxyethanol, and mild to moderate degeneration was observed in rats treated with 1500 ppm. Moderate to marked testicular degeneration was present in rats treated with 10,000 or 20,000 ppm 2-ethoxyethanol but not in rats treated with 5000 ppm. After 30 and 56 days of recovery from treatment with these chemicals, only partial recovery from testicular degeneration was observed. There was no testicular degeneration after 60 days of treatment with 1500 to 6000 ppm 2-butoxyethanol. 2-Methoxyethanol treatment for 13 weeks resulted in a progressive anemia associated with a cellular depletion of bone marrow and fibrosis of the splenic capsule. Anemia was also seen with 2-ethoxyethanol, but evidence of an adaptive response was indicated by increased hematopoiesis in the bone marrow, spleen, and liver. Toxicity with 2-butoxyethanol was limited to the liver and hematopoietic system. Cytoplasmic alteration and a minimal hepatocellular degeneration were present in the liver of male and female rats. A minimal anemia was present, and a hematopoietic response was evident in the bone marrow and spleen. In mice, 2-methoxyethanol and 2-ethoxyethanol had similar effects on the testes, spleen, and adrenal gland (females only). A dose-related degeneration of the germinal epithelium in seminiferous tubules of the testes was more severe with 2-methoxyethanol than with 2-ethoxyethanol. A dose-related increase in splenic hematopoiesis was also more prominent with 2-methoxyethanol. Both 2-methoxyethanol and 2-ethoxyethanol caused a prominent lipid vacuolization of the X-zone of the adrenal gland in female mice. There were no chemical-related lesions attributed to 2-butoxyethanol administration in mice. All three of the glycol ethers were negative in Salmonella typhimurium mutation tests conducted with and without induced hamster and rat liver S9. In the mouse lymphoma L5178Y cell mutation assay, 2-ethoxyethanol was negative without S9 but was weakly positive in the presence of induced rat liver S9; 2-methoxyethanol and 2-butoxyethanol were not tested in this assay. At high concentrations, 2-ethoxyethanol induced sister chromatid exchanges (SCEs) in Chinese hamster ovary cells with and without S9. Chromosomal aberrations (Abs) were also induced by 2-ethoxyethanol, but only in the absence of S9 and without a delay in cell cycle. In contrast, 2-butoxyethanol induced cell cycle delay but did not induce SCEs or Abs with or without S9. 2-Ethoxyethanol was the only glycol ether tested for induction of sex-linked recessive lethal mutations in germ cells of Drosophila melanogaster; both feeding and injection trials were negative. In summary, based on survival, decreased body weight gains, and histopathologic effects, the rank order of toxicity for the three glycol alkyl ethers was 2-methoxyethanol>2-ethoxyethanol>2-butoxyethanol; the toxic effects were more severe in rats than in mice. In the 13-week study of 2-methoxyethanol in rats, a no-observed-adverse-effect level (NOAEL) was not reached, since testicular degeneration in males and decreased thymus weights in males and females occurred at the lowest concentration administered (750 ppm). In the 13-week study of 2-ethoxyethanol in rats, the NOAEL for decreased thymus weights in males was 1250 ppm; for female rats treated with 2-ethoxyethanol for 13 weeks, the NOAEL for all histopathologic and hematologic effects was 5000 ppm. In rats treated with 2-butoxyethanol for 13 weeks, the NOAEL for liver degeneration was 1500 ppm in males and females. For male mice treated with 2-methoxyethanol for 13 weeks, the NOAEL for testicular degeneration and increased hematopoiesis in the spleen was 2000 ppm. A NOAEL was not reached for female mice treated with 2-methoxyethanol, since adrenal gland hypertrophy and increased hematopoiesis in the spleen occurred at the lowest concentration administered (2000 ppm). For male mice treated with 2-ethoxyethanol for 13 weeks, the NOAEL for testicular degeneration and increased hematopoiesis in the spleen was 20,000 ppm. For female mice in the 13-week study of 2-ethoxyethanol, the NOAEL for adrenal gland hypertrophy and increased hematopoiesis in the spleen was 5000 ppm. No clear chemical-related effects were seen in male or female mice administered 2-butoxyethanol for 13 weeks at concentrations as high as 6000 ppm. Synonyms: 2-Methoxyethanol: Ethylene glycol monomethyl ether; methyl cellosolve; 2-Ethoxyethanol: Ethylene glycol monoethyl ether; cellosolve; 2-Butoxyethanol: Ethylene glycol monobutyl ether; butyl cellosolve.

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