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PubMed2006

Evaluation of whether accelerated protein evolution in chordates has occurred before, after, or simultaneously with gene duplication.

Gene duplication and loss are predicted to be at least of the order of the substitution rate and are key contributors to the development of novel gene function and overall genome evolution. Although it has been established that proteins evolve more rapidly after gene duplication, we were interested in testing to what extent this reflects causation or association. Therefore, we investigated the rate of evolution prior to gene duplication in chordates. Two patterns emerged; firstly, branches, which are both preceded by a duplication and followed by a duplication, display an elevated rate of amino acid replacement. This is reflected in the ratio of nonsynonymous to synonymous substitution (mean nonsynonymous to synonymous nucleotide substitution rate ratio [Ka:Ks]) of 0.44 compared with branches preceded by and followed by a speciation (mean Ka:Ks of 0.23). The observed patterns suggest that there can be simultaneous alteration in the selection pressures on both gene duplication and amino acid replacement, which may be consistent with co-occurring increases in positive selection, or alternatively with concurrent relaxation of purifying selection. The pattern is largely, but perhaps not completely, explained by the existence of certain families that have elevated rates of both gene duplication and amino acid replacement. Secondly, we observed accelerated amino acid replacement prior to duplication (mean Ka:Ks for postspeciation preduplication branches was 0.27). In some cases, this could reflect adaptive changes in protein function precipitating a gene duplication event. In conclusion, the circumstances surrounding the birth of new proteins may frequently involve a simultaneous change in selection pressures on both gene-copy number and amino acid replacement. More precise modeling of the relative importance of preduplication, postduplication, and simultaneous amino acid replacement will require larger and denser genomic data sets from multiple species, allowing simultaneous estimation of lineage-specific fluctuations in mutation rates and adaptive constraints.

Amino Acid Substitution↗
PubMed2002

Different types and rates of genome evolution detected by comparative sequence analysis of orthologous segments from four cereal genomes.

Orthologous regions in barley, rice, sorghum, and wheat were studied by bacterial artificial chromosome sequence analysis. General microcolinearity was observed for the four shared genes in this region. However, three genic rearrangements were observed. First, the rice region contains a cluster of 48 predicted small nucleolar RNA genes, but the comparable region from sorghum contains no homologous loci. Second, gene 2 was inverted in the barley lineage by an apparent unequal recombination after the ancestors of barley and wheat diverged, 11-15 million years ago (mya). Third, gene 4 underwent direct tandem duplication in a common ancestor of barley and wheat 29-41 mya. All four of the shared genes show the same synonymous substitution rate, but nonsynonymous substitution rates show significant variations between genes 4a and 4b, suggesting that gene 4b was largely released from the strong purifying selection that acts on gene 4a in both barley and wheat. Intergenic retrotransposon blocks, many of them organized as nested insertions, mostly account for the lower gene density of the barley and wheat regions. All but two of the retrotransposons were found in the regions between genes, while all but 2 of the 51 inverted repeat transposable elements were found as insertions in genic regions and outside the retrotransposon blocks.

Biological Evolution↗
PubMed1994

Molecular analysis of rRNA and cholera toxin genes carried by the new epidemic strain of toxigenic Vibrio cholerae O139 synonym Bengal.

Vibrio cholerae O139 synonym Bengal recently caused large epidemics of cholera-like disease in Bangladesh and India. We compared the restriction fragment length polymorphisms of ctxA and rRNA genes (ribotypes) in 27 isolates of V. cholerae O139 from patients in Bangladesh and India with those of 48 isolates of V. cholerae O1 from patients and 21 V. cholerae isolates from surface waters in Bangladesh, which included 2 O139 and 19 other non-O1 isolates. Ribotyping of the isolates with BglI revealed that all 29 isolates of O139 vibrios belonged to a single ribotype, suggesting a clonal nature of the infection. However, the O139 vibrios comprised two ctxA genotypes and carried three or more copies of the ctxA gene, and the chromosomal locations of these copies were unlike those of the El Tor or classical vibrios. Analysis of the restriction fragment length polymorphisms of the rRNA genes suggested that V. cholerae O139 isolates are more closely related to El Tor strains of V. cholerae O1 than were 19 other non-O1 vibrios and 33 classical V. cholerae O1 isolates that were studied. However, further studies are needed to determine whether V. cholerae O139 originated from mutations and genetic changes in a V. cholerae O1 strain or was due to the acquisition of virulence genes by a previously unknown V. cholerae non-O1 strain.

Bacterial Typing Techniques↗
PubMed1994

NTP Comparative Toxicology Studies of Corn Oil, Safflower Oil, and Tricaprylin (CAS Nos. 8001-30-7, 8001-23-8, and 538-23-8) in Male F344/N Rats as Vehicles for Gavage.

The types and levels of fats in the diet are known to affect the incidence of certain neoplasms in humans and rodents. In long-term toxicity and carcinogenicity studies in rodents, the level of dietary fat is altered by using oil as a vehicle to administer unpalatable or volatile chemicals. Control male rats receiving a corn oil vehicle have a higher incidence of pancreatic proliferative lesions and a lower incidence of mononuclear cell leukemia than untreated control males. Therefore, the National Toxicology Program (NTP) designed studies to evaluate the role of several oils in altering cancer rates in male rats. The NTP study reported here was part of a larger program that included cooperative agreements with Dartmouth Medical School, Northwestern Medical School, and the University of Missouri. The program was designed to study the mechanisms by which corn oil induces pancreatic cancer. To evaluate corn oil as well as two other gavage vehicles for potential toxicity, corn oil, safflower oil, and tricaprylin were administered by gavage to male F344/N rats for 2 years. The rats that received corn oil were also made available to the university investigators for study of the corn oil-induced pancreatic lesions. Each vehicle was administered by gavage at volumes of 2.5, 5, or 10 mL/kg body weight once daily for 5 days per week. In the corn oil study, a control of 10 mL saline/kg was also included. To evaluate the potential role of corn oil in promoting a pancreatic proliferative effect, 500 mg dichloromethane/kg body weight was administered in 2.5, 5, or 10 mL corn oil/kg body weight for 2 years to male F344/N rats. Dichloromethane was chosen because the chemical appeared to cause pancreatic proliferative lesions when administered by gavage in a corn oil vehicle but not when the exposure was by inhalation. In each of these studies, the term "dose" refers to the volume of gavage vehicle administered. 2-YEAR STUDIES OF CORN OIL, SAFFLOWER OIL, AND TRICAPRYLIN: Survival and Body Weights: Two-year survival was increased in male rats receiving corn oil (untreated control, 26/50; saline control, 32/50; 2.5 mL/kg, 33/50; 5 mL/kg, 38/50; 10 mL/kg, 40/50) primarily due to a dose-related decreased incidence of mononuclear cell leukemia. The mean body weights of all dosed groups were at least 5% higher than those of the untreated and saline controls by week 48, but the mean body weights of groups receiving 2.5 or 5 mL corn oil/kg decreased during the final weeks of the study (after week 89) and were similar to those of the controls at the end of the study. Two-year survival was slightly increased in male rats receiving safflower oil relative to that of the controls (untreated control, 30/50; 2.5 mL/kg, 33/50; 5 mL/kg, 40/50; 10 mL/kg, 36/50). The mean body weight of male rats receiving 10 mL safflower oil/kg was at least 5% greater than that of the controls after week 45 and was 16% greater by the end of the study. Two-year survival of high-dose tricaprylin males was lower than that of the controls (untreated control, 31/50; 2.5 mL/kg, 30/50; 5 mL/kg, 31/50; 10 mL/kg, 23/53) due to moribund kills and deaths that appeared to be related to toxicity. The mean body weight of the high-dose group was lower than that of the controls throughout the study, although the difference was less than 5% after week 61. Pathology Findings: In the corn oil study, there were significant dose-related increased incidences of pancreatic exocrine hyperplasia and adenoma (hyperplasia: 8/50, 28/47, 28/50, 35/50; adenoma: 1/50, 8/47, 10/50, 23/50; carcinoma: 0/50, 0/47, 1/50, 0/50 in the untreated control, 2.5, 5, and 10 mL/kg groups, respectively). The incidence and severity of nephropathy decreased with dose (incidence [mean severity grade]: 47/50 [2.1], 43/48 [1.8], 45/50 [1.4], 40/49 [1.2]). The incidences of pheochromocytomas (benign, malignant, or complex) of the adrenal medulla were also decreased in dosed rats (23/49, 21/50, 5/50, 9/50). The incidence of mononuclear cell leukemia was significantly decreased in rats dosed with corn oil (27/50, 16/50, 11h corn oil (27/50, 16/50, 11/50, 7/50). In rats receiving safflower oil, the incidences of pancreatic exocrine hyperplasia and adenoma increased significantly with dose (hyperplasia: 8/50, 14/50, 29/49, 30/50; adenoma: 1/50, 7/50,15/49, 28/50; carcinoma: 0/50, 0/50, 0/49, 1/50 in the untreated control, 2.5, 5, and 10 mL/kg groups, respectively). There was a decrease in the severity, but not in the incidence, of nephropathy, a common lesion in aging F344/N rats (incidence [mean severity grade]: 49/50 [2.0], 50/50 [1.8], 47/50 [1.1], 49/49 [1.1]). There were decreased incidences of mononuclear cell leukemia (33/50, 19/50, 18/50, 7/51). In the tricaprylin study, there were significant dose-related increased incidences of pancreatic exocrine hyperplasia and adenoma (hyperplasia: 8/49, 9/49, 18/49, 28/50; adenoma: 2/49, 6/49,13/49,18/50 in the untreated control, 2.5, 5, and 10 mL/kg groups, respectively). The incidence of proliferative lesions of the forestomach increased significantly with dose (basal cell hyperplasia: 4/50, 7/50, 12/49, 21/52; squamous cell papilloma: 0/50, 0/50, 3/50, 10/53). The incidence of nephropathy was significantly decreased in high-dose rats, and the severity of nephropathy decreased with dose (incidence [mean severity grade]: 46/50 [2.0], 42/50 [1.5], 45/50 [1.7], 27/49 [0.9]). In high-dose rats, the incidence of mononuclear cell leukemia was decreased (23/50, 28/50, 22/50, 9/53). 2-YEAR STUDY OF DICHLOROMETHANE IN CORN OIL: Survival and Body Weights: Two-year survival increased slightly with dose in the three groups receiving 500 mg dichloromethane/kg in 2.5, 5, or 10 mL corn oil/kg (23/50, 28/50, 31/50) due to a dose-related decrease in the incidence of mononuclear cell leukemia. The rats receiving 500 mg dichloromethane/kg without corn oil were sacrificed within the first 3 weeks of the study due to the severe toxicity of dichloromethane. The final mean body weight of the high-dose rats was greater than the final mean body weights of groups receiving dichloromethane in 2.5 or 5 mL corn oil/kg. Pathology Findings: There was a dose-related increase in the incidence of pancreatic proliferative exocrine lesions in rats receiving dichloromethane in 2.5, 5, and 10 mL corn oil/kg (hyperplasia: 28/50, 38/50, 44/50; adenoma: 9/50,19/50, 41/50; carcinoma: 0/50,1/50, 3/50). The incidences of pancreatic exocrine hyperplasia and adenoma in rats receiving dichloromethane in 5 or 10 mL, but not 2.5 mL, corn oil were increased compared to the incidences in rats receiving comparable volumes of corn oil alone (hyperplasia: 2.5 mL, 28/47; 5 mL, 28/50;10 mL, 35/50; adenoma: 8/47,10/50, 23/50; carcinoma: 0/47,1/50, 0/50). There were significantly increased incidences of pituitary gland pars distalis adenoma in rats receiving dichloromethane in corn oil (20/50, 18/49, 16/49) when compared to those in rats receiving comparable volumes of corn oil alone (10/50, 6/49, 7/50). The incidence of mammary gland adenoma and fibroadenoma (combined) was significantly increased in rats receiving dichloromethane in 10 mL corn oil/kg (7/50) when compared to rats receiving dichloromethane in 2.5 mL corn oil/kg (1/50), but was not significantly increased when compared to the group receiving 10 mL of corn oil alone (3/50). The incidences of mammary gland adenoma and fibroadenoma (combined) were 7/50 for the untreated safflower oil controls and 6/50 for the untreated tricaprylin controls. The incidence of mononuclear cell leukemia decreased in the group receiving dichloromethane in 10 mL corn oil/kg (13/50,14/50, 5/50). GENETIC TOXICOLOGY: Neither safflower oil nor corn oil was mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535, with or without S9. Tricaprylin, in contrast, was mutagenic in strain TA1535 with, but not without, S9. Tricaprylin did not induce mutations in strains TA97, TA98, or TA100, with or without S9. SUMMARY: These studies were designed to evaluate the effects of various concentrations of an oil very high in polyunsaturated fat (safflower oil), an oil containing high levels of polyunsaturated and monounsaturated fats (corn oil), and an oil containing saturated medium-chain fatty acids (tricaprylin) on the incidence and pattern of neoplasms in the F344/N rat. In addition, safflower oil and tricaprylin were evaluated as replacements for the corn oil vehicle. These studies demonstrate that safflower oil and tricaprylin do not offer significant advantages over corn oil as a gavage vehicle in long-term rodent studies. Corn oil, safflower oil, and tricaprylin each caused hyperplasia and adenoma of the exocrine pancreas, decreased incidences of mononuclear cell leukemia, and reduced incidences or severity of nephropathy in male F344/N rats. There was an increased incidence of squamous cell papillomas of the forestomach in F344/N rats receiving 10 mL tricaprylin/kg. Further, the use of corn oil as a gavage vehicle may have a confounding effect on the interpretation of chemical-induced proliferative lesions of the exocrine pancreas and mononuclear cell leukemia in male F344/N rats. Synonyms: Corn Oil - Maize oil, Maydol Tricaprylin - Trioctanoin; 1,2,3-trioctanoyl glycerol; Glycerol trioctanoate

Journal Article↗
PubMed1992

Fetal recruitment of anthropoid gamma-globin genes. Findings from phylogenetic analyses involving the 5'-flanking sequences of the psi gamma 1 globin gene of spider monkey Ateles geoffroyi.

We determined the nucleotide sequence of a 2.5 kb DNA fragment (1 kb is 10(3) base-pairs) that includes exon 1, intron 1 and about 1.4 kb of 5'-flanking DNA of the spider monkey gamma 1-globin pseudogene locus and compared this sequence to its homologous from other primates and rabbit. This region of the gamma 1 locus of spider monkey still retains conserved regulatory elements, suggesting that it became a pseudogene late in New World monkey phylogeny. In the 250 base-pair region immediately 5' from the transcription start site where many known regulatory elements are located, a higher rate of nucleotide substitutions occurred in the ancestral anthropoid (human, ape and monkey) lineage than in the prosimian (galago) lineage, as was also the case for non-synonymous substitutions in the coding region. The opposite pattern was observed for most other non-coding regions and for synonymous substitutions. These substitution patterns correlate with the embryonic-to-fetal transformation of the gamma-globin genes of the ancestral anthropoids. Analysis of the 5'-flanking sequences suggests that 11 gene conversion events have occurred in the anthropoid gamma-gene lineages. In the parts of the 5'-flanking region where no gene conversions have been detected, gamma 2-gene sequences have accumulated more nucleotide changes than gamma 1, which suggests that the gamma 2 gene was the more redundant duplicate that may have accumulated first the nucleotide changes responsible for the anthropoid fetal pattern of gamma-globin gene expression.

Animals↗
PubMed2004

The ambush hypothesis: hidden stop codons prevent off-frame gene reading.

Coding sequences lack stop codons, but many stops appear off-frame. Off-frame stops (stops in -1 and +1 shifted reading frames, termed hidden stops) terminate frame-shifted translation, potentially decreasing energy, and resource waste on nonfunctional proteins. Benefits may include reduced waste elimination costs and avoidance of potentially cytotoxic frame-shifted products. Our "ambush" hypothesis suggests that hidden stops are sometimes selected for. Codons of many amino acids can contribute to hidden stops, depending on the synonymous position state and adjacent codons. In vertebrate mitochondria, 31.75% of all amino acid combinations can form hidden stops. Codons with more potential to form hidden stops have greater usage frequency and bias in their favor among synonymous codons. Among primates, predicted mitochondrial rRNA secondary structure stability correlates negatively with the number of hidden stops in the mitochondrial genome. The taxonomic distribution of genetic codes suggests that +1 frameshifts might be more frequent than -1 frameshifts. This is confirmed by analyses of primate mitochondrial genomes: species with unstable rRNAs have more +1 stops, but the correlation is weak for -1 stops. High hidden stop density seems to be an adaptation in species with slippage prone ribosomes (unstable rRNAs). Hidden stops may thus compensate for reduced efficiency of some parts of the biosynthetic machinery. Some experimental data confirm our hypothesis: gene expression increases with the experimentally manipulated number of stops in the promoter region of a gene, suggesting biotechnological applications.

Animals↗
PubMed1998

Higher frequency of concerted evolutionary events in rodents than in man at the polyubiquitin gene VNTR locus.

The polyubiquitin gene is an evolutionarily conserved eukaryotic gene, encoding tandemly repeated multiple ubiquitins, and is considered to be subject to concerted evolution. Here, we present the nucleotide sequences of new alleles of the polyubiquitin gene UbC in humans and CHUB2 in Chinese hamster, which encode a different number of ubiquitin units from those of previously reported genes. And we analyze the concerted evolution of these genes on the basis of their orthologous relationship. That the mean of the synonymous sequence difference Ks which is defined as the number of synonymous substitution relative to the total number of synonymous sites, within the UbC and CHUB2 genes (0.192 +/- 0.096) is significantly less than Ks between these genes (0.602 +/- 0.057) provides direct evidence for concerted evolution. Moreover, it also appears that concerted evolutionary events have been much more frequent in CHUB2 than in UbC, because Ks within CHUB2 (0.022 +/- 0.018) is much less than that within UbC (0.362 +/- 0.192). By a numerical simulation, postulating that the major mechanism of concerted evolution in polyubiquitin genes is unequal crossing over, we estimated the frequency of concerted evolutionary events of CHUB2 at 3.3 x 10(-5) per year and that of UbC at no more than 5.0 x 10(-7) per year.

Amino Acid Sequence↗
PubMed2004

Frequent false detection of positive selection by the likelihood method with branch-site models.

Positive Darwinian selection promotes fixations of advantageous mutations during gene evolution and is probably responsible for most adaptations. Detecting positive selection at the DNA sequence level is of substantial interest because such information provides significant insights into possible functional alterations during gene evolution as well as important nucleotide substitutions involved in adaptation. Efficient detection of positive selection, however, has been difficult because selection often operates on only a few sites in a short period of evolutionary time. A likelihood-based method with branch-site models was recently introduced to overcome such difficulties. Here I examine the accuracy of the method using computer simulation. I find that the method detects positive selection in 20%-70% of cases when the DNA sequences are generated by computer simulation under no positive selection. Although the frequency of such false detection varies depending on, among other things, the tree topology, branch length, and selection scheme, the branch-site likelihood method generally gives misleading results. Thus, detection of positive selection by this method alone is unreliable. This unreliability may have resulted from its over-sensitivity to violations of assumptions made in the method, such as certain distributions of selective strength among sites and equal transition/transversion ratios for synonymous and nonsynonymous substitutions.

Adaptation, Biological↗
PubMed1986

NTP Toxicology and Carcinogenesis Studies of Chlorpheniramine Maleate (CAS No. 113-92-8) in F344/N Rats and B6C3F1 Mice (Gavage Studies).

Toxicology and carcinogenesis studies of chlorpheniramine maleate (99% pure), a widely used antihistaminic drug in human and veterinary medicine, were conducted by administering this chemical in deionized water by gavage to groups of 50 male and 50 female F344/N rats and B6C3F1 mice, 5 days per week for 103 weeks. The doses used were: male rats-- 0, 15, or 30 mg/kg; female rats-- 0, 30, or 60 mg/kg; male mice-- 0, 25, or 50 mg/kg; female mice-- 0, 100, or 200 mg/kg. The selection of these doses was based largely on data from 14-day or 16-day studies and 13-week studies in which reduced body weight gain and reduced survival occurred at higher doses. Doses used in the 2-week studies ranged from 40 to 640 mg/kg in rats and 25 to 800 mg/kg in mice; in the 13-week studies, doses ranged from 3.75 to 60 mg/kg in rats and 12.5 to 200 mg/kg in mice. The recommended human adult daily oral dose of chlorpheniramine maleate is up to 0.32 mg/kg. Doses originally selected for male mice in the 2-year study were 0, 100, or 200 mg/kg; however, because of poor survival, that study was stopped and a new study was started at doses of 0, 25, or 50 mg/kg. At the termination of the study (week 104), survival of high dose female rats (6/50) and high dose male mice (15/50) was lower than that of the vehicle controls (29/50 and 39/50, respectively). Survival of all other dosed groups was comparable to that of respective vehicle control groups. Mean body weights of dosed rats were about 10%-15% (male) or about 10%-25% (female) lower than those of vehicle controls; mean body weights of female mice were generally 20%-35% lower than those of vehicle controls. No compound-related gross or microscopic pathologic effects were observed in either species in the 16-day or 13-week studies. Hyperactivity and hyperexcitability associated with dosing were frequently noted in the 13-week and 2-year studies. There were no significant positive trends or increases in the incidences of neoplasms in either male or female rats dosed with chlorpheniramine maleate for 103 weeks. Marginal increases in the incidences of adrenal gland capsule adenomas in male mice (vehicle control, 2/50; low dose, 7/49; high dose, 4/49) were not considered to be compound related, since there was not a corresponding increase in the incidence of adrenal gland capsule hyperplasia (46/50; 33/49; 22/49). A positive trend was seen for subcutaneous tissue tumors in male mice (4/50; 5/49; 8/50); this marginal effect was not considered to be compound related. The incidences of thyroid gland follicular cell cysts (2/48; 10/49; 13/47), thyroid gland follicular cell hyperplasia (3/48; 29/49; 36/47), and thyroid gland follicular cell adenomas (0/48; 4/49; 2/47) were greater in dosed female mice than in vehicle controls. This finding is toxicologically important,since thyroid gland neoplasms are uncommon in mice and are often preceded by hyperplasia of the follicular epithelium. The major route of excretion of chlorpheniramine or its metabolites is in the urine. In male F344 rats orally administered 14C-chlorpheniramine maleate at doses of 2 or 20 mg/kg, there was essentially no difference in the percentage of urinary or fecal excretion of radioactivity between these dose levels. Chlorpheniramine maleate was not mutagenic to Salmonella strains TA98, TA100, TA1535, or T1537 in the presence or absence of S9 metabolic activation systems prepared from the liver of Aroclor 1254-treated male Sprague-Dawley rats or male Syrian hamsters. Chlorpheniramine maleate did not induce forward mutations at the TK locus of L5178Y mouse lymphoma cells with or without metabolic activation. In Chinese hamster ovary cells in culture, chlorpheniramine maleate induced a weak but reproducible increase in sister-chromatid exchanges in the absence of exogenous metabolic activation. Chromosomal aberrations were induced at the highest dose tested but only in the presence of S9 from Aroclor 1254-induced Sprague-Dawley male rat liver. An audit of the experimental data was conducted for these 2-year carcinogenesis studies on chlorpher these 2-year carcinogenesis studies on chlorpheniramine maleate. No data discrepancies were found that influenced the final interpretations. Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenicity for F344/N rats or B6C3F1 mice of either sex administered chlorpheniramine maleate in deionized water, 5 days per week for 2 years. Due to high mortality in high dose female rats and high dose male mice, the sensitivity of these groups to detect a carcinogenic response was reduced. Chlorpheniramine maleate had a proliferative effect in the thyroid gland of female mice, as shown by the increased incidences of follicular cell cysts and hyperplasia in both low dose and high dose groups. Synonyms: 2-p-chloro-a-(2-dimethylaminoethyl) benzyl]pyridine maleate; 2-Pyridinepropanamine; gamma-[4-chlorophenyl]-N,N-dimethyl-[z]-2-butenedioate Trade Names: Allerclor; Allergisan; Antagonate; Chlormene; Chlorprophenpyridamine maleate; Chlor-Trimeton; Chlor-Tripolon; Chloropiril; C-Meton; Histadur; Histaspan; Lorphen; M.P. Chlorcaps T.D.; Piriton; Pyridamal-100; Teldrin

Journal Article↗
PubMed2006

SNPeffect v2.0: a new step in investigating the molecular phenotypic effects of human non-synonymous SNPs.

UNLABELLED: Single nucleotide polymorphisms (SNPs) constitute the most fundamental type of genetic variation in human populations. About 75 000 of these reported variations cause an amino acid change in the translated protein. An important goal in genomic research is to understand how this variability affects protein function, and whether or not particular SNPs are associated to disease susceptibility. Accordingly, the SNPeffect database uses sequence- and structure-based bioinformatics tools to predict the effect of non-synonymous SNPs on the molecular phenotype of proteins. SNPeffect analyses the effect of SNPs on three categories of functional properties: (1) structural and thermodynamic properties affecting protein dynamics and stability (2) the integrity of functional and binding sites and (3) changes in posttranslational processing and cellular localization of proteins. The search interface of the database can be used to search specifically for polymorphisms that are predicted to cause a change in one of these properties. Now based on the Ensembl human databases, the SNPeffect database has been remodeled to better fit an automatically updatable structure. The current edition holds the molecular phenotype of 74 567 nsSNPs in 23 426 proteins. AVAILABILITY: SNPeffect can be accessed through http://snpeffect.vib.be.

Algorithms↗
PubMed2005

A PYY Q62P variant linked to human obesity.

Peptide YY (PYY) has been implicated in the control of food intake through functional studies in rodents and humans. To investigate whether genetic alterations within this gene result in abnormal weight in humans, we sequenced its coding exons and splice sites in a large cohort of extremely obese [n = 379; average body mass index (BMI), 49.0 kg/m2] and lean (n = 378; average BMI, 19.5 kg/m2) individuals. In total, three rare non-synonymous variants were identified, only one of which, PYY Q62P, exhibited familial segregation with body mass. Through serendipity, previous studies based on cell culture revealed this precise variant to have altered receptor-binding selectivity in vitro. We further show, using mouse peptide injection experiments, that while the wild-type PYY peptide reduces food intake, the mutant PYY 62P had an insignificant effect in reducing food intake in vivo. Taken together, these results are the first to support that rare sequence variants within PYY can influence human susceptibility to obesity.

Amino Acid Sequence↗
PubMed2005

[Study of sequence variations of Epstein-Barr virus LMP1 gene in nasopharyngeal carcinoma].

OBJECTIVE: To detect the sequence variations frequently found within the N- and C-terminal regions of Epstein-Barr virus (EBV) LMP1 gene in nasopharyngeal carcinoma (NPC) and to study the underlying mechanisms. METHODS: Fresh tumor tissues were sampled from 63 patients with untreated NPC encountered in Affiliated Tumor Hospital of Sun Yat-sen University, Guangzhou. The N-terminal region of EBV LMP1 gene was amplified with nested polymerase chain reaction (PCR), followed by XhoI enzyme digestion. Nested PCR was also employed to detect the 30 base pairs deletion within the C-terminal region. Four-colored fluorescence terminator sequencing method was applied for bi-directional solid-phase sequencing of the 8 representative PCR products in 4 cases of NPC. The DNA sequence within the N- and C-terminal regions of LMP1 gene was then analyzed. RESULTS: There were 4 patterns of sequence variations, namely, wt-XhoI/wt-LMP1 (4 cases, 6.3%), wt-XhoI and XhoI-loss/del-LMP1 (4 cases, 6.3%), wt-XhoI/del-LMP1 (5 cases, 7.9%) and XhoI-loss/del-LMP1 (50 cases, 79.5%), detected in the 63 studied cases. Sequence analysis showed that the EBV LMP1 gene had underwent non-synonymous and synonymous substitutions, as compared with the prototype of B95-8 cells. The ratio of non-synonymous to synonymous substitutions was 2.25. CONCLUSIONS: XhoI-loss/del-LMP1 is the predominant sequence variation pattern of EBV LMP1 gene in NPC from Guangzhou. The XhoI-loss variation seems to develop on top of del-LMP1. When compared with the EBV LMP1 gene in peripheral blood B-lymphocytes of virus carriers and in preinvasive epithelial lesions (reported previously), it is likely that the sequence variation patterns of LMP1 gene may represent 4 different phases of intrahost evolution of EBV during nasopharyngeal carcinogenesis.

Adult↗
PubMed1986

NTP Toxicology and Carcinogenesis Studies of Dimethylvinyl Chloride (1-Chloro-2-Methylpropene) (CAS No. 513-37-1) in F344/N Rats and B6C3F1 Mice (Gavage Studies).

Dimethylvinyl chloride is a clear colorless liquid, which, because of its volatility and flammability at room temperature, is a significant fire hazard. It has a boiling point of 68.1 degrees C (155 degrees F) and a density at 20 degrees C of 0.919 g/ml. Dimethylvinyl chloride is a byproduct in the production of 3-chloro-2-methylpropene by the chlorination of isobutene. It is not known to be produced in the United States for other than laboratory purposes. This chemical was nominated for toxicologic studies because of its reported presence in ambient air in the Baltimore area and was selected for toxicologic characterization because of its structural similarity to the known animal and human carcinogen, vinyl chloride monomer. Toxicology and carcinogenesis studies of dimethylvinyl chloride (96%-98% pure), a structural analog of vinyl chloride monomer, a known human carcinogen, by administered dimethylvinyl chloride in corn oil by gavage to groups of 50 male and 50 female F344/N rats and B6C3F1 mice at doses of 0, 100, or 200 mg/kg body weight 5 days per week for 102 or 103 weeks. The selection of these doses was based on results of 13-week studies, which included depression of body weight at doses of 500 mg/kg or above in rats as well as histopathologic changes intestinal epithelium, bone marrow, hepatocytes, and the testes at doses of 250 mg/kg and above; doses in mice were selected on the basis of histopathologic changes in lymphopoietic cells, liver, pancreatic islets, ovary, testis, and spleen, with changes being most prominent at doses of 500 mg/kg and above. In the 2-year studies, body weights of rats and mice given 100 mg/kg were comparable to those of the vehicle controls except for the last few weeks in mice when body weights were markedly lower than those for the vehicle controls. At 200 mg/kg, the mean body weights of rats and mice were progressively decreased relative to those of vehicle controls, with the significant departure from vehicle controls occurring somewhat earlier in males than in females. Survival of vehicle control rats and mice was comparable to historical values; however, survival of dosed male and female rats was significantly lower than that of vehicle controls, with the incidence of mortality being more severe at the high dose than at the low dose. There were no survivors in the high dose group of male rats after week 85 or in the high dose group of female rats after week 97. Survival was significantly lower among dosed male and female mice compared with vehicle controls. In the absence of toxicological findings that would explain the early deaths, it is assumed that the high incidence of tumors and chemical-related toxicity contributed to the decreased survival of dosed rats and mice. In rats, the severity and incidence of nonneoplastic lesions were minimal; these lesions included necrosis of the duodenum and epithelial hyperplasia at the sites of tumor formation--the nasal cavity, esophagus, and forestomach. In mice, the severity of nonneoplastic lesions was also minimal; the lesions included necrosis of the liver, bone marrow granulocytic hyperplasia, and inflammation of the nasal cavity (small number, females only.) Several types of neoplastic lesions occurred with significantly increased incidences in dosed animals as shown in the following table (see page 11 of Technical Report). Among rats, these lesions included malignant epithelial tumors of the nasal cavity and squamous cell tumors of the oral cavity, esophagus, and forestomach in males and females. The increased number of fibroadenomas of the mammary gland in female rats may have been related to dimethylvinyl chloride administration. The lack of a clear dose-response relationship for certain tumors in rats is considered to be related to the increased number of early deaths observed in the high dose groups. Among dosed mice, there were significantly increased incidences of squamous cell carcinomas of the forestomach (both sexes), squamous cell papillomas of the forestomach (males), and squamous cell carcinomas of the preputial gla preputial gland (males). The increased incidence of papillary adenomas of the harderian gland and alveolar/bronchiolar adenomas or carcinomas in female mice may have been related to administration of dimethylvinyl chloride. Limited metabolism studies of 14C-labeled dimethylvinyl chloride were conducted in male F344/N rats and B6C3F1 mice. Single doses of 150 mg/kg were administered to rats for 1, 2, or 4 consecutive days. About 25% of the administered doses was exhaled as carbon dioxide; this amount was independent of the number of doses administered. Another 25%-35% of the administered dose was exhaled; 96% of this parent was material. Approximately 35% and 6% were excreted in the urine and feces, respectively. The elimination half-life of radioactive label was 3-4 days for the liver and kidney, the two organs containing the greatest amounts of the administered dose. In mice, a much smaller fraction of the dose was exhaled and a larger proportion was excreted in urine compared with rats. Dimethylvinyl chloride was not mutagenic in four strains ofSalmonella typhimurium with or without metabolic activation, but it was mutagenic in the mouse lymphoma L5178Y/TK± assay in the absence of metabolic activation. Sister-chromatid exchanges were induced in Chinese hamster ovary cells with and without metabolic activation, but there was no increase in chromosomal aberrations. When fed to Drosophila, dimethylvinyl chloride induced significant increases in the frequencies of both sex-linked recessive lethal mutations and reciprocal translocations. Studies of the immunotoxicity of dimethylvinyl chloride were conducted in which female B6C3F1 mice received daily oral doses of 0, 50, 100, 200, or 400 mg dimethylvinyl chloride per kilogram body weight. Compound-related increases in susceptibility to bacterial infection and decreases in macrophage cytostasis were observed at all doses. At the highest dose, the decreased resistance to bacterial and viral challenge could be related to alterations in specific immune function. However, the increased mortality in rats and mice in the 2-year studies was not relatable to infectious processes. An audit of the experimental data was conducted for these2-year toxicology and carcinogenesis studies on dimethylvinyl chloride. No data discrepancies were found that influenced the final interpretations. Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenicity of dimethylvinyl chloride for both sexes of F344/N rats and B6C3F1 mice. This was based on increased incidences of neoplasms of the nasal cavity, oral cavity, esophagus, and forestomach of male and female F344/N rats. B6C3F1 mice showed increased incidences of squamous cell neoplasms of the forestomach in males and females and squamous cell carcinomas of the preputial gland in males. Synonym: 1-chloro-2-methylpropene

Journal Article↗
PubMed1993

NTP Toxicology and Carcinogenesis Studies of 3,4-Dihydrocoumarin (CAS No. 119-84-6) in F344/N Rats and B6C3F1 Mice (Gavage Studies).

3,4-Dihydrocoumarin was nominated by the Food and Drug Administration and the National Cancer Institute for study because of its widespread use as a flavoring agent in beverages, gelatins, puddings, candy, and other food items; as a fragrance in perfumes, creams, and cosmetics; and because of interest in the structure-activity relationships of the coumarin derivatives. Toxicity and carcinogenicity studies were conducted by administering 3,4-dihydrocoumarin (99% pure) in corn oil by gavage to groups of male and female F344/N rats and B6C3F1 mice for 16 days, 13 weeks, and 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, and peripheral blood cells of mice. 16-DAY STUDY IN RATS: Groups of five male and five female rats received 3,4-dihydrocoumarin in corn oil by gavage at doses of 0, 190, 375, 750, 1,500, or 3,000 mg/kg body weight 5 days per week for a total of 12 doses in a 16-day period. All male and female rats given 3,000 mg/kg, and four male rats and five female rats given 1,500 mg/kg died. Body weight gains and final mean body weights of rats receiving 190, 375, or 750 mg/kg were similar to those of the controls. There were no clinical findings of organ-specific toxicity or evidence of impaired blood coagulation. 16-DAY STUDY IN MICE: Groups of five male and five female mice received 3,4-dihydrocoumarin in corn oil by gavage at doses of 0, 140, 280, 560, 1,125, or 2,250 mg/kg body weight 5 days per week for a total of 12 doses in a 16-day period. All mice given 2,250 mg/kg died. Body weight gains and final mean body weights of mice receiving 140, 280, 560, and 1,125 mg/kg were similar to those of the controls. There were no clinical findings of organ-specific toxicity or evidence of impaired blood coagulation. 13-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats received 3,4-dihydrocoumarin in corn oil by gavage at doses of 0, 75, 150, 300, 600, or 1,200 mg/kg body weight 5 days per week for 13 weeks. Two male rats and five female rats given 1,200 mg/kg died. The body weight gain and final mean body weight of male rats that received 1,200 mg/kg were significantly lower than those of the controls, but the final mean body weights of other dosed groups of male rats and all dosed groups of female rats were similar to or slightly greater than those of the controls. Platelet counts were significantly lower in males and females receiving 600 and 1,200 mg/kg and in females receiving 300 mg/kg. Hemoglobin and hematocrit values and erythrocyte counts were significantly lower in males that received 300 mg/kg or more. The absolute and relative liver and kidney weights of males and females receiving 600 and 1,200 mg/kg were significantly greater than those of the controls. Hepatocellular hypertrophy was observed in rats given 300, 600, and 1,200 mg/kg. The high dose selected for the 2-year study was 600 mg/kg, which was below the level at which mortality, lower final mean body weights, and treatment-related liver lesions were observed. 13-WEEK STUDY IN MICE: Groups of 10 male and 10 female mice received 3,4-dihydrocoumarin in corn oil by gavage at doses of 0, 100, 200, 400, 800, or 1,600 mg/kg body weight 5 days per week for 13 weeks. Eight male and five female mice receiving 1,600 mg/kg died. Deaths in other groups were attributed to dosing accidents. Final mean body weights of dosed male and female mice were similar to those of the controls, and there were no treatment-related changes in any hematologic parameters. The absolute and relative liver weights of males and females that received 1,600 mg/kg and the relative kidney weight of males that received 1,600 mg/kg were significantly greater than those of the controls. No treatment-related lesions were noted. The high dose selected for the 2-year study was 600 mg/kg, which was below the level at which mortality, lower final mean body weights, and treatment-related liver lesions were observed. 2-YEAR STUDY IN RATS: Groups of 60 male and 60 female rats received 3,4-dihydrocoumarin in corn oil by gavage at age at doses of 0, 150, 300, or 600 mg/kg body weight. After 15 months, up to 10 animals from each group were evaluated. Survival, Body Weights, and Clinical Findings: Survival rates of dosed male rats were lower than that of the controls (O mg/kg, 28/51; 150 mg/kg, 12/50; 300 mg/kg, 8/50; 600 mg/kg, 2/50) but survival rates of dosed female rats were similar to that of the controls (31/50, 21/51, 26/50, 23/51). The decreased survival in dosed male rats was attributed to a chemical-related increase in the severity of nephropathy. The final mean body weight of male rats receiving 600 mg/kg was lower than that of the controls, but the final mean body weights of other dosed groups of male rats and all dosed groups of female rats were similar to those of the controls. No clinical findings related to chemical administration were observed. Hematology and Clinical Chemistry: At the 15-month interim evaluation, the hemoglobin concentrations, mean erythrocyte volumes, or mean erythrocyte hemoglobin concentrations in the 300 and 600 mg/kg female rats were slightly, but significantly, lower than those of the controls. In males, only the hemoglobin concentration in the 600 mg/kg group was significantly lower. Serum levels of alkaline phosphatase, alanine aminotransferase, sorbitol dehydrogenase, or g-glutamyltransferase in the 300 and 600 mg/kg male rats were significantly higher than those in the controls. In females, alkaline phosphatase and g-glutamyltransferase levels were significantly higher in the 600 mg/kg group. Pathology Findings: The principal lesions associated with the administration of 3,4-dihydrocoumarin to rats occurred in the kidney and forestomach. There was a chemical related increase in the severity of nephropathy in all dosed male rats and in 300 and 600 mg/kg female rats. There was a corresponding increased incidence of parathyroid gland hyperplasia, probably as a result of compromised renal function. In the standard evaluation of single kidney sections, renal tubule adenomas were observed in one 150 and two 600 mg/kg males and one each in the control, 150, and 300 mg/kg females. Transitional cell carcinomas were also observed in two 600 mg/kg male rats. However, an extended evaluation of step sections identified significantly higher incidences of focal hyperplasia and adenoma in the 600 mg/kg males than in controls (hyperplasia: 0/50, 5/48, 6/47, 8/50; adenoma: 1/50,1/48, 3/47, 6/50). The incidence of forestomach ulcers in all groups of dosed male rats was significantly greater than that of the controls (4/47, 14/48, 20/50, 16/46). STOP-EXPOSURE EVALUATION: A group of 40 male rats received 600 mg/kg 3,4-dihydrocoumarin in corn oil by gavage for 9 months, when 20 of the animals were necropsied and evaluated. The remainder of the male rats received only the corn oil vehicle until they died or until the end of the study. Similarly, a group of 30 male rats received 600 mg/kg 3,4-dihydrocoumarin in corn oil by gavage for 15 months, when 10 of the rats were necropsied and evaluated. The remaining 20 rats received only corn oil until the end of the study. A group of 20 vehicle control male rats was necropsied at 9 months, and another 10 vehicle control male rats were necropsied at 15 months. The severity of nephropathy in male rats of the stop-exposure groups was significantly greater than that of males examined at the 9- and 15-month interim evaluations. This was expected because nephropathy is a progressive degenerative disease that naturally increases in severity with age. 2-YEAR STUDY IN MICE: Groups of 70 male and 70 female mice received 3,4-dihydrocoumarin in corn oil by gavage at doses of 0, 200, 400, or 800 mg/kg body weight. After 15 months, five to 10 animals from each group were evaluated. Additional groups of 8 to 10 animals were evaluated for clinical pathology after 15 months. Survival, Body Weights, and Clinical Findings Survival rates of dosed male and female mice were similar to those of the controls (males: O mg/kg, 42/50; 200 mg/kg, 39/51; 400 mg/kg, 34/51; 800 mg/kg, 38/50; females: 36/51, 39/50, 41/50, 28/52). Final mean body weights of dosed male and female mice were similar to those of the controls. No clinical findings were noted that were related to chemical administration. Hematology and Clinical Chemistry: There were no differences in hematology or clinical chemistry parameters that were considered to be chemical related. Pathology Findings: The principal neoplasms associated with the administration of 3,4-dihydrocoumarin to mice occurred in the liver. There were significantly increased incidences of hepatocellular adenomas in all groups of dosed female mice. Further, the incidences of multiple hepatocellular adenomas in dosed female mice were greater than that of the controls (control, 0/51; 200 mg/kg, 6/50; 400 mg/kg, 9/50; 800 mg/kg, 9/52). However, there was no corresponding increased incidence of hepatocellular carcinoma in dosed female mice (3/51, 2/50, 4/50, 6/52), and the incidences of hepatocellular adenoma or carcinoma were similar between dosed and control male groups (adenoma: 29/50, 23/51, 36/51, 31/50; carcinoma: 11/50, 11/51, 11/51, 6/50). The incidence of alveolar/bronchiolar adenoma in the 200 and 400 mg/kg male mice was marginally greater than that of the controls (8/50,15/50,15/51,10/50). However, these neoplasms were not considered chemical related because the increased incidence was slight and there was no corresponding increased incidence in the 800 mg/kg group. The incidence of alveolar/bronchiolar neoplasms in female mice was similar between the dosed and control groups (adenoma: 2/51, 5/50, 1/48, 3/51; carcinoma: 0/51, 1/50, 0/48, 0/51). In the standard evaluation of single sections of kidney, focal hyperplasia and adenoma or carcinoma of the renal tubule were identified in several dosed male mice, but not in controls [adenoma or carcinoma (combined): 0/50,1/51, 2/51,1/49; hyperplasia: 2/50, 2/51, 5/51, 2/49]. In an extended evaluation of step sections, a few additional males with focal hyperplasia or renal tubule adenomas were identified in the dosed groups. However, the incidences of these lesions in dosed groups of male mice were not significantly greater than those of the controls, and did not increase with dose (hyperplasia: 0/50,1/51, 3/51, 1/49; renal tubule adenoma: 0/50, 0/51, 2/51, 1/49). Therefore, the low number of renal tubule neoplasms in male mice was not considered to be chemical related. GENETIC TOXICOLOGY: 3,4-Dihydrocoumarin did not induce gene mutations in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 with or without exogenous metabolic activation (S9). It induced sister chromatid exchanges but not chromosomal aberrations in cultured Chinese hamster ovary cells, with and without S9. No induction of micronuclei was noted in peripheral blood erythrocyte samples obtained from male and female B6C3F1 mice at the end of the 13-week toxicology study. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was some evidence of carcinogenic activity of 3,4-dihydrocoumarin in male F344/N rats based on increased incidences of renal tubule adenomas and focal hyperplasia. The transitional cell carcinomas in two 600 mg/kg males may also have been chemical related. There was no evidence of carcinogenic activity of 3,4-dihydrocoumarin in female F344/N rats receiving 150, 300, or 600 mg/kg. There was no evidence of carcinogenic activity of 3,4-dihydrocoumarin in male B6C3F1 mice receiving 200, 400, or 800 mg/kg. There was some evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined). 3,4-Dihydrocoumarin caused ulcers, hyperplasia, and inflammation of the forestomach, parathyroid gland hyperplasia, and increased severity of nephropathy in male rats. Synonyms: 1,2-benzodihydropyrone, 2H-1-benzopyran-2-one, 2-chromanone, 3,4-dihydro-2H-1-benzopyran-2-one, dihydrocoumarin, hydrocoumarin, o-hydroycinnamic acid, delta-lactone-hydrocinnamic acid, melilotin, melilotine, melilotol, 2-oxochroman

Journal Article↗
PubMed1992

NTP Technical report on the toxicity studies of ortho-, meta-, and para- Nitrotoluenes (CAS Nos. 88-72-2, 99-08-1, 99-99-0) Administered in Dosed Feed to F344/N Rats And B6C3F1 Mice.

Nitrotoluenes are high production volume chemicals used in the synthesis of agricultural and rubber chemicals and in various dyes. Because of differences in the metabolism of the 3 isomers and their capability to bind to DNA, comparative toxicity studies of o-, m-, or p-nitrotoluene were conducted in F344 rats and B6C3F1 mice. Animals were evaluated for histopathology, clinical pathology, and toxicity to the reproductive system. The nitrotoluenes were also studied in several in vitro and in vivo assays for genetic toxicity. In 14-day studies, o-nitrotoluene, m-nitrotoluene, or p-nitrotoluene was administered in the feed to male and female rats and mice at concentrations ranging from 388 to 20000 ppm (5 animals/chemical/species/sex/dose). There were no effects on survival or clinical signs of toxicity in these studies, although animals at the higher doses showed decreases in body weight gains relative to controls. In the 13-week studies, o-, m-, or p-nitrotoluene was given to male and female rats and mice (10 animals/chemical/species/ sex/dose) in the feed at concentrations between 625 and 10000 ppm. The estimated daily doses based on measures of feed consumption were 40 to 900 mg nitrotoluene/kg body weight/day for rats and 100 to 2000 mg/kg/day for mice and were similar for each of the 3 isomers when compared for each dietary level/sex/species. There were no effects on survival in any of the studies, and clinical signs of toxicity were limited to decreases in feed consumption. Decreased body weight gains occurred in dosed rats and mice in all studies at the higher dose levels and were most pronounced in rats receiving o-nitrotoluene. In rats, histopathologic analyses after 13 weeks of dosing showed toxicity to kidney, spleen, and testis in animals receiving any of the 3 isomers, and toxicity to the liver and mesothelium in male rats given o-nitrotoluene. Kidney toxicity observed in male rats was characterized by the presence of hyaline droplets in tubular epithelial cells, attributed to an increase in the level of alpha-2μ-globulin. Pigment, possibly lipofuscin, and karyomegaly in the p-nitrotoluene study were present in the renal tubular epithelium of dosed male and female rats. In the spleen of treated male and female rats, there was a mild increase in hematopoiesis, hemosiderin deposition, and/or congestion; this effect was most severe with the para-isomer, followed by the ortho- and then the meta-isomer. Administration of o-, m-, or p-nitrotoluene impaired testicular function of the rat, shown by degeneration of the testis and reduction in sperm concentration, motility, and spermatid number. All 3 isomers increased the length of the estrous cycle in rats. Hepatic toxicity was characterized by cytoplasmic vacuolization and oval cell hyperplasia and by an increase in the level of serum bile acids, SDH, and ALT activities in male rats given o-nitrotoluene. There was no histopathologic evidence for liver toxicity in male or female rats with the m- or p-isomers, or in female rats with the o-isomer; but evidence of liver injury was observed in these groups, indicated by increases in relative liver weights and elevations in bile acids and liver enzymes in serum. Mesotheliomas of the tunica vaginalis were observed in 3/10 male rats receiving o-nitrotoluene at 5000 ppm, and mesothelial cell hyperplasia was observed in 2/10 male rats receiving o-nitrotoluene at 10000 ppm. The only histopathologic evidence for toxicity in mice in the 13- week studies occurred in the olfactory epithelium in mice receiving o-nitrotoluene, where the chemical caused degeneration and metaplasia. No liver lesions were noted in mice, but the 3 isomers caused increases in relative liver weights. There was no toxicity to the reproductive system in male or female mice treated with any of the nitrotoluene isomers. The 3 nitrotoluene isomers were not mutagenic in Salmonella typhimurium strains TA100, TA1535, TA1537, and TA98. Only p-nitrotoluene induced chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells, and this required metabolic activation. Sister-chromatid exchanges were increased in CHO cells following exposure to each isomer; the requirement for metabolic activation varied. Only p-nitrotoluene was studied in the mouse lymphoma L5178Y test; it caused mutations with metabolic activation. Unscheduled DNA synthesis (UDS) was increased in in vitro incubations of hepatocytes isolated from both sexes of rats and mice after receiving a single in vivo oral dose of o-nitrotoluene. UDS was not increased in a similar study with male rats given m- or p-nitrotoluene. o-Nitrotoluene also induced s-phase DNA synthesis in hepatocytes of rats but not in those of mice. In summary, the 3 nitrotoluene isomers were toxic to the kidney, spleen and/or reproductive system in rats; o-nitrotoluene also caused lesions in the liver of male rats. No treatment-related lesions were noted in mice except with o-nitrotoluene where olfactory epithelium degeneration occurred. The increase in relative liver weights and the increase in UDS in liver indicate that all 3 isomers affected the liver of female rats and of male and female mice, even though histopathologic lesions were not observed. In general, the extent of the toxicity was most severe with the o-isomer in both rats and mice. o-Nitrotoluene was carcinogenic in male rats in 13-week studies, based on the occurrence of mesothelioma and mesothelial cell hyperplasia in dosed groups. Synonyms: o-NT, 2NT, 2-nitrotoluene, 2-methylnitrobenzene, 2-nitrotoluol; m-NT, 3NT, 3-nitrotoluene, 3-methylnitrobenzene, 3-nitrotoluol; p-NT, 4NT, 4-nitrotoluene, 4-methylnitrobenzene, 4-nitrotoluol.

Journal Article↗
PubMed1993

Toxicology and Carcinogenesis Studies of Furan (CAS No. 110-00-9) in F344 Rats and B6C3F1 Mice(Gavage Studies).

Furan serves as an intermediate in the synthesis and preparation of numerous linear polymers used to prepare temperature-resistant structural laminates and to prepare copolymers used in machine dishwashing products as alternatives to phosphorus- and nitrogen-containing detergents. Toxicology and carcinogenesis studies were conducted by administering furan (purity > 99%) in corn oil by gavage to groups of F344/N rats and B6C3F1 mice of each sex for 16 days, 13 weeks, and 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Drosophila melanogaster, mouse bone marrow cells, mouse L5178Y lymphoma cells, and Chinese hamster ovary cells. 16-Day Studies: Groups of five male rats received doses of 0, 5, 10, 20, 40, or 80 mg of furan per kg of body weight and groups of five female rats and five mice of each sex received doses of 0, 10, 20, 40, 80, and 160 mg/kg in corn oil by gavage. All male and female mice and female rats that received 160 mg/kg, all male and female rats and all male and four female mice that received 80 mg/kg, and three male mice that received 40 mg/kg died by day 8. Final mean body weights of male rats that received 20 mg/kg and of male and female rats that received 40 mg/kg were significantly lower than controls. Final mean body weights of male mice that received 10 or 20 mg/kg were significantly greater than controls. Mottled and enlarged livers were observed at necropsy in male rats that received 20, 40, or 80 mg/kg and in females that received 40, 80, or 160 mg/kg. No lesions were observed at necropsy that were considered related to furan administration in mice. 13-Week Studies: Groups of 10 rats of each sex and groups of 10 female mice received doses of 0, 4, 8, 15, 30, or 60 mg of furan per kg of body weight, and groups of 10 male mice received doses of 0, 2, 4, 8, 15, or 30 mg/kg in corn oil by gavage. Nine male and four female rats that received 60 mg/kg died before the end of the studies. There were no chemical-related deaths in mice. Final mean body weights of male rats that received 15 or 30 mg/kg and female rats that received 60 mg/kg were significantly lower than controls. Final mean body weights of male mice that received 60 mg/kg were significantly lower than controls. Relative and absolute liver weights in both sexes of rats and mice were increased in groups that received furan, as were relative and absolute kidney weights in female rats that received furan. Thymus weights were decreased in all groups of rats that received furan. Toxic lesions of the liver (bile duct hyperplasia, cholangiofibrosis, cytomegaly and degeneration of hepatocytes, and nodular hyperplasia of hepatocytes) were associated with furan administration in all dose groups of rats; the severity of the lesions increased with dose. Kidney lesions (tubule dilatation and necrosis of tubule epithelium) were present in rats that received 30 or 60 mg/kg. Thymic atrophy and testicular or ovarian atrophy were also observed in rats exposed to 60 mg/kg furan. Toxic liver lesions (cytomegaly, degeneration, and necrosis of hepatocytes) were also present in all groups of furan-exposed mice. Bile duct hyperplasia and cholangiofibrosis were observed in groups of mice receiving 30 or 60 mg/kg. Doses selected for the 2-year studies of rats and mice were based on the hepatotoxicity associated with exposure to furan. 2-Year Studies: Groups of 70 rats of each sex were administered 2, 4, or 8 mg furan per kg body weight in corn oil by gavage 5 days per week for 2 years. After 9 and 15 months of chemical exposure, 10 rats per group were evaluated for the presence of treatment-associated lesions. Groups of 50 mice of each sex received doses of 8 or 15 mg/kg furan 5 days per week for 2 years. Body Weight and Survival: Mean body weights of male rats that received 8 mg/kg furan were lower than controls from approximately week 73 to the end of the study. Survival of male and female rats that received 8 mg/kg was lower than controls from approximately week 85 to the end of the studies as a result of moribund condition associatedassociated with liver and biliary tract neoplasms and mononuclear cell leukemia. Mean body weights of male and female mice that received 15 mg/kg furan were lower than controls during the studies. Survival of low- and high-dose male and high-dose female mice was lower than controls from approximately week 80 to the end of the studies as a result of moribund condition associated with liver neoplasms. Neoplastic and Nonneoplastic Lesions: Cholangiocarcinoma of the liver occurred in all groups of dosed rats (males: control, 0/50; low dose, 43/50; mid dose, 48/50; high dose, 49/50; females: 0/50; 49/50; 50/50; 48/50) and was present in many rats of each sex at the 9- and 15-month interim evaluations (9-month: males - 0/10, 5/10, 7/10, 10/10; females - 0/10, 4/10, 9/10, 10/10; 15-month: males - 0/10, 7/10, 9/10, 6/10; females - 0/10, 9/10, 9/10, 7/10). Hepatocellular adenomas or carcinomas (combined) were significantly increased in male rats after 2 years of chemical administration (1/50, 5/50, 22/50, 35/50) and hepatocellular adenomas were significantly increased in female rats (0/50, 2/50, 4/50, 7/50); hepatocellular neoplasms were not observed at the 9- or 15-month interim evaluations. Increased incidences of numerous nonneoplastic liver lesions were present in rats administered furan. These lesions included biliary tract fibrosis, hyperplasia, chronic inflammation, and proliferation and hepatocyte cytomegaly, cytoplasmic vacuolization, degeneration, nodular hyperplasia, and necrosis. The incidence of mononuclear cell leukemia was increased in male and female rats that received 4 or 8 mg/kg furan (males: 8/50, 11/50, 17/50, 25/50; females: 8/50, 9/50, 17/50, 21/50); the incidence in the 8 mg/kg groups of each sex exceeded the historical control ranges for corn oil gavage studies. The severity of nephropathy increased with dose and the incidence was significantly increased in all groups of dosed rats; this increased severity was accompanied by an associated increased incidence of parathyroid hyperplasia (renal secondary hyperparathyroidism). The incidence of forestomach hyperplasia was increased in male and female rats (males: 1/50, 4/49, 7/50, 6/50; females: 0/50, 2/50, 5/50, 5/50) and the incidence of subacute inflammation of the forestomach was increased in female rats (0/50, 1/50, 5/50, 6/50). No forestomach neoplasms were observed in males; a squamous papilloma was present in one low-dose female. The incidences of hepatocellular adenomas and carcinomas were significantly increased in mice receiving furan (males: adenoma - 20/50, 33/50, 42/50; carcinoma - 7/50, 32/50, 34/50; females: adenoma - 5/50, 31/50, 48/50; carcinoma - 2/50, 7/50, 27/50). The incidences of numerous nonneoplastic hepatocellular lesions were increased in dosed mice. These lesions included hepatocyte cytomegaly, degeneration, necrosis, multifocal hyperplasia, and cytoplasmic vacuolization and biliary tract dilatation, fibrosis, hyperplasia, and inflammation. The incidences of benign pheochromocytoma and focal hyperplasia of the adrenal medulla were increased in low- and high-dose male and in high-dose female mice (benign pheochromocytoma: males - 1/49, 6/50, 10/50; females - 2/50, 1/50, 6/50). The incidences of squamous papilloma, focal inflammation, and papillary hyperplasia of the forestomach were increased in male mice (squamous papilloma: 0/49, 1/50, 3/50; focal inflammation: 9/49, 13/50, 21/50; papillary hyperplasia: 7/49, 14/50, 22/50). Stop-Exposure Study: A separate 2-year study was conducted in which 50 male rats were administered 30 mg/kg furan in corn oil by gavage 5 days per week for 13 weeks and then maintained for the remainder of the 2 years without additional furan administration. Groups of 10 animals were evaluated for the presence of treatment-related lesions at the end of the 13-week period of furan administration and at 9 and 15 months. Neoplastic and Nonneoplastic Lesions: Cholangiocarcinoma of the liver occurred with an overall incidence of 100% (40/40) and hepatocellular carcinoma occurred with an overall incidence of 15% (6/40) in stop-exposure male rats that survived at least 9 months. Cholangiocarcinoma was observed in all 10 males at both the 9-month and 15-month interim evaluations. Hepatocellular carcinoma was first observed in 2 males at the 15-month interim evaluation. Genetic Toxicology: Furan was negative for induction of gene mutations in Salmonella typhimurium strains TA100, TA1535, TA1537, and TA98 in the presence and the absence of exogenous metabolic activation (S9). Furan was negative for the induction of sex-linked recessive lethal mutations in germ cells of male Drosophila melanogaster when administered either by feeding or by injection. In vitro tests for genotoxicity in mammalian cells, however, were positive. Furan induced trifluorothymidine resistance in mouse L5178Y lymphoma cells in the absence of S9, and sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells, with and without S9. Furan administered to male B6C3F1 mice by intraperitoneal injection induced chromosomal aberrations but not sister chromatid exchanges in bone marrow cells. Conclusions: Under the conditions of these 2-year gavage studies there was clear evidence of carcinogenic activity of furan in male and female F344/N rats based on increased incidences of cholangiocarcinoma and hepatocellular neoplasms of the liver and on increased incidences of mononuclear cell leukemia. There was clear evidence of carcinogenic activity of furan in male and female B6C3F1 mice based on increased incidences of hepatocellular neoplasms of the liver and benign pheochromocytomas of the adrenal gland. Nonneoplastic liver lesions associated with furan administration in rats and mice included biliary tract fibrosis, hyperplasia, inflammation, and proliferation, as well as hepatocellular cytomegaly, degeneration, hyperplasia, necrosis, and vacuolization. In rats, increased severity of nephropathy with an associated increased incidence of parathyroid hyperplasia was associated with exposure to furan. Synonyms: Divinylene oxide, tetrole, furfuran, oxole, 1,4-epoxy-1,3-butadiene, axole, oxacyclopentadiene

Journal Article↗
PubMed2002

Effect of tandem rare codon substitution and vector-host combinations on the expression of the EBV gp110 C-terminal domain in Escherichia coli.

Gp110 of Epstein-Barr virus (EBV) is a glycoprotein that functions exclusively during the assembly of EBV nucleocapsid and the release of infectious EBV. Its C-terminal tail domain (gp110 CTD) is essential for gp110's function and may provide signals that are responsible for the assembly and release of EBV. In the present study, to get large amounts of gp110 CTD for structural analysis, the effects of vector system, codon usage, and host strain on expression levels of gp110 CTD in Escherichia coli have been investigated. The coding region of gp110 CTD (11 kDa) was subcloned into the expression vectors pSE 280, pET-15b, pET-29a, pMAL-c2x, and pGEX-4T-1. Except the pMAL-c2x construct, all the others failed to express detectable amounts of recombinant gp110 CTD. Substituting a tandem rare AGA (Arg) codon with a synonymous CGC (Arg) codon facilitated expression of the recombinant protein, while a protease-deficient host E. coli strain helped in the accumulation of a soluble form of gp110 CTD fusion. The secondary structures of the obtained recombinant gp110 CTD purified from soluble extracts and inclusion bodies were compared using circular dichroism analysis. In aqueous solutions, both samples equally adopt a mixed alpha-helix and beta-sheet conformation as well as a partly unordered structure. Notably, in the membrane-mimicking environments the helical propensity of gp110 CTD increased up to the previously predicted level based on its sequence, suggesting that gp110 CTD may fold into a more stable conformation through interactions with the cell membrane.

Circular Dichroism↗
PubMed2003

Invertebrate species with nonpelagic larvae have elevated levels of nonsynonymous substitutions and reduced nucleotide diversities.

Under a nearly neutral model in which most amino acid substitutions are slightly deleterious, variation in demography, population structure, and other ecological factors among closely related species can potentially modify the effective population size or the selective regime, leading to differences in the rate of nonsynonymous substitution. Ratios of nonsynonymous to synonymous substitutions (d(N)/d(S)) between species were analyzed in a sea star genus (Patiriella) and a molluscan genus (Littorina), each with diverse modes of reproduction, including multiple lineages with pelagic and nonpelagic larvae. In both genera, lineages with nonpelagic larvae had significantly higher d(N)/d(S) ratios than lineages with pelagic larvae. The hypothesis that the elevated d(N)/d(S) ratios in species with nonpelagic larvae was due to reduced effective population size was tested by comparing nucleotide diversities in three genera of gastropod mollusks (Littorina, Crepidula, and Hydrobia), each with several modes of reproduction. Overall, there was a significant (p < 0.05) reduction in nucleotide diversity in species with nonpelagic larvae compared to species with pelagic larvae.

Animals↗