Biomedical subjects

N P Lang

Publications and source records attributed to N P Lang.

At least 19 recordsLinked to original sources

PubMed2000

Quantitative analysis of interindividual variation of glutathione S-transferase expression in human pancreas and the ambiguity of correlating genotype with phenotype.

Analysis of glutathione S-transferases (GSTs) of the alpha, mu, and pi classes by reverse-phase high-performance liquid chromatography and electrospray-ionization mass spectrometry in 43 samples of normal human pancreas demonstrated a wide variation in expression of subunits P1, A1, A2, A4, M1, M2, and M3 and the presence of a novel form designated GST "A5." GSTA2 consisted of three forms that were differentially expressed between individuals in a manner consistent with allelic polymorphism at the hGSTA2 locus. Expression, in terms of microg GST subunit/mg cytosolic protein, varied by 6-15-fold for subunits P1, A2, and M3 and 17-30-fold in the case of GSTs A1 and M2. Less consistently expressed were GSTs M1a, M1b, A4, and A5. Among these, GSTM1 expression (excluding M1-null samples) varied 12-fold between samples, whereas GST A4 and A5 expression varied approximately 50-100-fold between samples, well beyond the range of other subunits, suggesting that their expression is highly inducible. Linear correlations (P < 0.001-0.003) existed between levels of the most consistently expressed GST, GSTP1, and total GSTs, GSTA2 and M3, and in GSTM1-positive samples, between GSTM1, M3, and P1. The correlation between GST subunits P1 and M3 was bimodal according to M1 genotype, reflecting the presence of the regulatory element in hGSTM3*B that is linked with the hGSTM1*A genotype. It is concluded that although a degree of regulation of expression of GSTs occurs in human pancreas, the variability of phenotype is high and might obscure the effects of genetic polymorphisms on individual cancer susceptibility. Interindividual variation of GST expression is, therefore, a factor that should be taken account of in epidemiological studies.

Adolescent↗

PubMed2000

Persistence patterns of Porphyromonas gingivalis, Prevotella intermedia/nigrescens, and Actinobacillus actinomyetemcomitans after mechanical therapy of periodontal disease.

BACKGROUND: The aim of this study was to determine the distribution patterns of Porphyromonas gingivalis, Prevotella intermedia/nigrescens, and Actinobacillus actinomycetemcomitans in periodontitis patients after standard mechanical periodontal therapy, and to determine factors increasing the odds to detect these target organisms in treated sites. METHODS: Eight hundred fifty-two (852) separate subgingival microbial samples were taken from the mesial and distal aspects of every tooth in 17 patients. Target organisms were identified culturally. RESULTS: The 3 microorganisms showed different persistence patterns: P. gingivalis was detected in a high percentage of subjects (59%), but in a low proportion of sites (5.4%). P. intermedia/nigrescens was detected in all subjects except one, and in 40.6% of the tested sites. Only 5 subjects were A. actinomycetemcomitans positive, but 2 of them showed a very high number of positive sites (44% and 75%, respectively). A highly significant relationship was found between a subject's tendency to bleed upon sampling and the number of P. intermedia/nigrescens-positive sites. A significant portion of the variation in frequency of persisting P. gingivalis could be explained by the frequency of persisting pockets deeper than 4 mm. No similar relationship could be established between clinical parameters and A. actinomycetemcomitans. On a site level, the odds of detecting P. gingivalis increased by a factor of 2.47 (P= 0.0001) for every millimeter of residual probing depth; the odds of detecting P. intermedia/nigrescens increased by a factor of 1.84 (P= 0.0001). CONCLUSIONS: If, after standard mechanical periodontal therapy, a large number of sites continue to bleed, one may expect an increased number of sites positive for P. intermedia/ nigrescens. If many deep pockets persist, a greater number of P. gingivalis-positive sites can be expected.

Adult↗

PubMed1999

Macromolecular adduct formation and metabolism of heterocyclic amines in humans and rodents at low doses.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are heterocyclic amines formed during the cooking of meat and fish. Both are genotoxic in a number of test systems and are carcinogenic in rats and mice. Human exposure to these compounds via dietary sources has been estimated to be under 1 microg/kg body wt. per day, although most laboratory animal studies have been conducted at doses in excess of 10 mg/kg body wt. per day. We are using accelerator mass spectrometry (AMS), a tool for measuring isotopes with attomole sensitivity, to study the dosimetry of protein and DNA adduct formation by low doses of MeIQx and PhIP in rodents and comparing the adduct levels to those formed in humans. The results of these studies show: 1, protein and DNA adduct levels in rodents are dose-dependent; 2, adduct levels in human tissues and blood are generally greater than in rodents administered equivalent doses; and 3, metabolite profiles differ substantially between humans and rodents for both MeIQx and PhIP, with more N-hydroxylation (bioactivation) and less ring oxidation (detoxification) in humans. These data suggest that rodent models do not accurately represent the human response to heterocyclic amine exposure.

Animals↗

PubMed1999

In vivo human metabolism of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

To better understand the interactions of the pathways of activation and detoxification on the metabolism of the putative carcinogen, PhIP, we administered a dose of 70-84 microg [2-14C] PhIP (17.5 [microCi 14C) 48-72 h before scheduled colon surgery. Blood and urine collected for the next 48-72 h was evaluated by linear accelerator mass spectroscopy (AMS) and scintillation counting LC-MS to identify specific PhIP metabolites. The thermostable phenol sulfotransferase (SULT1A1) phenotype was correlated with the 4'-PhIP-SO4 levels in the urine at 0-4 h (R = 0.86, P = 0.059). The CYP1A2 activity had a negative correlation with PhIP serum levels at 1 h (R = 0.94, P = 0.06) and a positive correlation with urine N-OH-PhIP levels at 0-4 h (R = 0.85, P = 0.15). This low level radioisotope method of determining the influence of phenotype on metabolism will significantly improve our understanding of the interrelationships of these pathways and provide a critical foundation for the development of individual risk assessment.

Adult↗

PubMed1999

Comparison of DNA adduct levels associated with exogenous and endogenous exposures in human pancreas in relation to metabolic genotype.

Recently, we examined normal human pancreas tissue for DNA adducts derived from either exogenous chemical exposure and/or endogenous agents. In an effort to explain the different types and levels of DNA adducts formed in the context of individual susceptibility to cancer, we have focused on gene-environment interactions. Here, we report on the levels of hydrophobic aromatic amines (AAs), specifically those derived from 4-aminobiphenyl (ABP), and DNA adducts associated with oxidative stress in human pancreas. Although these adducts have been reported in several human tissues by different laboratories, a comparison of the levels of these adducts in the same tissue samples has not been performed. Using the same DNA, the genotypes were determined for N-acetyltransferase 1 (NAT1), the glutathione S-transferase (GST) M1, GSTP1, GSTT1, and NAD(P)H quinone reductase-1 (NQO1) as possible modulators of adduct levels because their gene products are involved in the detoxification of AAs, lipid peroxidation products and in redox cycling. These results indicate that ABP-DNA adducts, malondialdehyde-DNA adducts, and 8-oxo-2'-deoxyguanosine (8-oxo-dG) adducts are present at similar levels. Of the metabolic genotypes examined, the presence of ABP-DNA adducts was strongly associated with the putative slow NAT1*4/*4 genotype, suggesting a role for this pathway in ABP detoxification.

Aminobiphenyl Compounds↗

PubMed1999

Attempts to obtain re-osseointegration following experimental peri-implantitis in dogs.

The purpose of this study was to examine the healing potential and re-osseointegration in peri-implant infection defects adjacent to various implant surfaces. In 7 female Beagle dogs, a total of 41 titanium oral implants (ITI, Straumann, Waldenburg; Switzerland) with a sink depth of 6 mm (diameter 2.8 mm) were placed transmucosally. Four different surface configurations (TPS: titanium plasma sprayed (10); SLA: sand blasted and acid-etched (13); M: machined and smooth (11); TPS furc.: titanium plasma sprayed with coronally placed perforation to mimic a furcation (7) were distributed among the animals and locations. Following a healing period of 3 months, silk ligatures were placed and oral cleaning procedures abolished for 4 months to induce a vertical bone loss of about 40%. Following mechanical and chemical cleansing (chlorhexidine and metronidazole) and disinfection, the lesions were either sham operated (11) or subjected to a GTR procedure using ePTFE (30). After 6 months of healing the animals were killed and the jaws histologically evaluated. Six membranes were lost TPS: (1); SLA: (2); M: (2); TPS furc: (1) and 3 membranes exposed TPS: (1); M: (2) and excluded from further evaluation. Owing to the loss of 1 implant and infection of the membranes in the TPS furc group, this implant configuration was discarded from further analysis. For TPS surfaces, bone fill was 2.6 mm (73% of the distance from the bottom of the defect to the shoulder of the implant) sites with (4 GTR) and 0.33 mm (14%) for sites without membrane (2 controls). Re-osseointegration was 0.5 mm (14%) in the test group and 0.3 mm (14%) in the control. For SLA surfaces bone fill was 2.3 mm (83%) for sites with (7 GTR) and 0.41 mm (15%) for sites without membranes (4 controls). Re-osseointegration was 0.6 mm (20%) and 0.3 mm (11%) respectively. Corresponding values for M surfaces were 2.2 mm (62%) with 4 GTR) and 0.82 mm (31%) without membranes. Re-osseointegration was 0.07 mm (2%) and 0.19 mm (7%) respectively. This study has documented that peri-implant infections defects may heal with bone fill provided that the infection is controlled through effective antibacterial therapy. However, true reosseointegration appears to be difficult to achieve.

Alveolar Process↗

PubMed1999

The development and validation of an occlusal site-specific plaque index to evaluate the effects of cleaning by tooth brushes and chewing gum.

These studies sought to develop and validate an occlusal site-specific plaque index to be used to measure plaque removal by brushing or chewing gum. The index divides the occlusal surfaces into imaginary zones from which scores are apportioned on a 0-4 basis dependent on the perceived % plaque coverage of each zone. Examiner calibration was conducted over 2 studies assessing inter-examiner reproducibility and intra-examiner repeatibility, respectively. Study 1 involved 2 examiners who recorded scores from the same 3 groups of subjects who had suspended tooth cleaning for 4 days. Analyses for inter-examiner reproducibility showed no significant mean differences between examiners or no significant differences between variances of the 2 examiners scores. Study 2 involved the same 2 examiners individually scoring 3 groups of subjects 2 x (approximately 60 min apart) for occlusal plaque. Analysis for intra-examiner repeatability showed no significant mean differences between the 2 scorings of each examiner. Furthermore, there were no significant differences between the variances of each examiner's scores except for 1 examiner in the repeatability exercise for the 1st group of subjects. Study 3 involved groups of subjects at 2 separate clinical sites (Bristol, England and Berne, Switzerland) being scored for occlusal plaque before and after toothbrushing with water or after no toothbrushing. Data from individual examiners and examiners combined revealed a significant reduction in occlusal plaque with brushing compared to no brushing. Study 4 was the same as study 3 but occlusal plaque was scored before and after chewing gum or not chewing gum. The Bristol examiner recorded a significant reduction in plaque by chewing gum compared to not chewing gum but the Berne examiner did not. The latter may have resulted from a considerable disparity in the number of evaluable occlusal surfaces between the two study sites. The index could be employed as part of the overall assessment or oral hygiene or used in clinical trials to study mechanical and chemical plaque control agents.

Adult↗

PubMed1999

Microbiological and clinical effects of an antiseptic dental varnish after mechanical periodontal therapy.

The aim of this study was to explore the microbiological and clinical effects of an antiseptic dental varnish when applied to periodontally diseased teeth after mechanical therapy. 20 subjects participated in this placebo controlled, double blind prospective longitudinal study. 2 experimental sites with a pocket probing depth > or =5 mm were chosen in each subject. The control varnish, consisting of ethanol, ethylacetate and polyvinylbutyral, was applied to one of the selected teeth and the test varnish, containing 1% chlorhexidine and 1% thymol in addition, was applied to the other one. Clinical parameters were assessed, and microbiological samples were obtained from the two study sites at baseline (6-10 weeks after completion of conventional periodontal therapy), and 2, 4 and 12 weeks thereafter. The mean PLI at baseline was very low and, therefore, only a minimal potential for a further improvement existed. During the 12-week observation period, the mean PLI increased significantly at sites treated with the placebo varnish, while no similar trend for an increase in PLI was detected in the test sites. The bleeding tendency seemed to remain unaffected by the application of the varnish. On the microbiological level, no relevant differences could be detected between placebo and test sites at baseline, or during the follow-up period. In conclusion, the application of a dental varnish with antimicrobial properties after mechanical periodontal therapy had little effect in subjects with good oral hygiene.

Adult↗

PubMed1999

Actinobacillus actinomycetemcomitans in Chinese adults. Serotype distribution and analysis of the leukotoxin gene promoter locus.

The aim of the present study was to examine the Actinobacillus actinomycetemcomitans carrier rate in Chinese subjects, and to determine serotype distribution, presence of the leukotoxin gene lktA and the structure of the lktA-promoter region. Subgingival microbiological samples were obtained from 31 Chinese subjects with moderate to advanced adult periodontitis, 73 young factory workers, and 81 adult residents of a rural area. Bacterial isolates phenotypically identified as A. actinomycetemcomitans were found in 116 of the 185 subjects (detection frequency over-all: 63%). Presence of the leukotoxin gene lktA was demonstrated for all 115 isolates that could be subcultured. The PCR analysis of the lktA-promoter region showed that none of these strains had the deletion in the promoter region known to enhance expression of lktA. No significant difference in the frequency of A. actinomycetemcomitans could be observed between the subjects of the 3 study groups. Analysis by logistic multiple regression indicated a homogeneous distribution of A. actinomycetemcomitans in the 3 cohorts and a lack of significant influence of subject gender or age. Serotype a was found in 21 subjects, serotype b was found in 9, serotype c in 67 and serotype e in 11 individuals. Serotype d was not detected in any subject. Nontypeable isolates, lacking serotype a, b, c, d, or e antigens, were found in 9 individuals. A high prevalence irrespective of gender, age, and cohort suggests that A. actinomycetemcomitans is a common constituent of the normal flora in the Chinese subjects of this study and suggests differences in the microbiological composition of subgingival plaque may exist for this population group as compared to north American and European populations.

Adolescent↗

PubMed1999

Clinical effects of root instrumentation using conventional steel or non-tooth substance removing plastic curettes during supportive periodontal therapy (SPT).

Although root instrumentation has been accepted as the most important cause-related treatment of periodontal diseases, repeated scaling and root planing may over time result in substantive loss of tooth substance and increased sensitivity of the teeth. In an effort to minimize these side effects of therapy, non-root substance removing curettes have been developed. However, the clinical effects of such plastic curettes with regard to the control of the periodontal infection has not yet been established. The aims of this study were, therefore, to compare the effects of root instrumentation using plastic curettes (Universal Perio Soft Scaler, Hawe-Neos Dental, Bioggio, TI, Switzerland) versus conventional steel curettes on the periodontal conditions during supportive periodontal therapy. 40 subjects participated in this parallel, randomized, double blind, prospective longitudinal clinical study following active peridontal therapy. 20 subjects served as a control group and were treated with conventional steel curettes during a supportive periodontal care visit (SPT). The other 20 subjects, the experimental group, were treated using plastic curettes during a similar SPT visit. Clinical parameters, such as bleeding on probing (BOP) and probing pocket depth (PPD), were assessed at baseline and 3-6 months later at the next regular SPT visit. In addition, the BOP percentage was determined 10 days following baseline. The results showed that there were no statistically significant differences between the 2 treatment modalities regarding BOP and PPD at any observation time. Both treatments were effective in reducing the BOP percentage which ranged from 17-42% at baseline by about 40% after 10 days (mean BOP baseline: 26%, mean BOP after 10 days: 16%). This clinical study suggests that non-root substance removing curettes may be valuable instruments for periodontally treated patients during maintenance care, thus minimizing trauma on the hard structures of the teeth.

Adult↗

PubMed1999

The identification of [2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans.

[2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), a putative human carcinogenic heterocyclic amine found in well-done cooked meat, was administered orally to three colon cancer patients undergoing a partial colonectomy. Forty-eight to seventy-two hours prior to surgery, subjects received a 70-84 microg dose of 14C. Urine and blood were analyzed by HPLC for PhIP and PhIP metabolites. Metabolites were identified based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis and susceptibility to enzymatic cleavage. In two subjects, approximately 90% of the administered [14C]PhIP dose was eliminated in the urine, whereas in the other, only 50% of the dose was found in the urine. One subject excreted three times more radioactivity in the first 4 h than did the others. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In all three subjects the most abundant urinary metabolite was identified as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-N2-glucuron ide (N-hydroxy-PhIP-N2-glucuronide), accounting for 47-60% of the recovered counts in 24 h. PhIP accounted for <1% of the excreted radiolabel in all three patients. Other metabolites detected in the urine at significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N3-glucuronide and PhIP-N2-glucuronide. In the plasma, N-hydroxy-PhIP-N2-glucuronide accounted for 60, 18 and 20% of the recovered plasma radioactivity at 1 h post PhIP dose in subjects 1, 2 and 3 respectively. Plasma PhIP was 56-17% of the recovered dose at 1 h post exposure. The relatively high concentration of N-hydroxy-PhIP-N2-glucuronide and the fact that it is an indicator of bioactivation make this metabolite a potential biomarker for PhIP exposure and activation. Determining the relative differences in PhIP metabolites among individuals will indicate metabolic differences that may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen.

Administration, Oral↗

PubMed1999

Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4, 5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a heterocyclic aromatic amine found in cooked meats and dietary exposure to PhIP has been implicated in the etiology of colon cancer in humans. PhIP, along with other heterocyclic aromatic amines, requires metabolic activation to exhibit genotoxic effects. PhIP is initially oxidized by the activity of cytochrome P4501A2 to produce 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a reaction occurring primarily in the liver. Whereas subsequent biotransformation of N-OH-PhIP via acetylation or sulfation can produce reactive electrophiles that readily bind to DNA, N-glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), functions as a detoxification mechanism. Although hepatic glucuronidation of N-OH-PhIP has been well characterized, the extrahepatic metabolism of this compound is poorly understood. Studies in our laboratory now indicate that the intestinal tract, and particularly the colon, is a significant site of glucuronidation of N-OH-PhIP. When assays were performed with microsomes prepared from the mucosa of the intestinal tract, it was determined that glucuronidation of N-OH-PhIP occurs throughout the intestinal tract, with activity approximately three times higher in the colon as that found in the upper intestine. Glucuronidation rates from colon microsomes showed considerable interindividual variability and incubation with N-OH-PhIP yielded two glucuronides. HPLC analysis showed that the predominant product formed is the N-OH-PhIP-N2-glucuronide, while the N3-glucuronide accounts for <10% of the total glucuronidation product. These rates approach the rates found in human liver microsomes, demonstrating the significance of extrahepatic metabolism of this food-borne carcinogen. Subsequent assays with human recombinant UGTs demonstrated that at least four human UGT isoforms, all from the UGT1A subfamily, are capable of catalyzing the biotransformation of N-OH-PhIP. Members of the UGT2B family available for this study did not conjugate N-OH-PhIP, although immunoinhibition studies in human liver microsomes strongly suggest the involvement of a UGT2B isoform(s) in this organ.

Animals↗

PubMed1999

Sulfating-activity and stability of cDNA-expressed allozymes of human phenol sulfotransferase, ST1A31 ((213)Arg) and ST1A32 ((213)His), both of which exist in Japanese as well as Caucasians.

We recently found single amino acid substitutions ((213)Arg/His and (223)Met/Val) in polymorphic human phenol-sulfating phenol sulfotransferase (SULT: cDNAs encoding ST1A3, P PST or HAST1/2) among Caucasians and African-Americans. In a Japanese population (n = 143), allele frequencies of (213)Arg and (213)His were 83.2 and 16. 8%, respectively, but the (223)Val allele was not found. (213)His homozygosity was reportedly associated with both very low (>7-fold) sulfating activities of p-nitrophenol (at 4 microM) and low thermostability in platelets. Sulfating-activity determinations using recombinant (213)Arg- and (213)His-forms (ST1A3*1 and ST1A3*2, respectively) did not, however, reveal appreciable deficiency in [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS)-dependent sulfation of p-nitrophenol (4 microM) by ST1A3*2 (7.5 vs. 10.2 nmol/min/nmol SULT for ST1A3). Kinetic parameters for p-nitrophenol for p-nitrophenol sulfation supported the slight decrease in sulfating activities at 4 microM (K(m), 0.82 vs. 1.75 microM; V(max), 13.2 vs. 13.1 nmol/min/nmol SULT, respectively, for ST1A3*1 and *2). p-Nitrophenyl sulfate-dependent 2-naphthol sulfation by ST1A3*2 was 69% of that by ST1A3*1 (p<0.05). However, ST1A3*2 was remarkably unstable at 45 and 37 degrees C as compared to ST1A3*1. The lower p-nitrophenol sulfating activity of ST1A3*2 may explain the lower platelet p-nitrophenol sulfation in ST1A3*2 homozygotes. Protein instability and ST1A3 gene regulation may be both involved in the polymorphism of p-nitrophenol sulfation in human tissues.

Arylsulfotransferase↗

PubMed1999

Education in periodontology. A need for a new teaching model.

The objectives of the present work were to elucidate faculty perceptions and effects on traditional teaching and the supplement of a problem-based learning model in periodontal education. Students and faculty members at one university were asked to respond to a battery of relevant questions in periodontology that had been previously discussed at two European academic workshops. Differences in responses were noticed both between faculty, students, and their responses as compared to responses given by participants at the European workshops. On several topics, faculty within the selected university held significantly different opinions. Thus lecture content and concepts may vary dependent on who gives the lecture and may not be consistent with the common interpretation of the scientific evidence. Responses given by those participating in the workshops indicated consensus. The study results suggest that students were able to extract appropriate conclusions from the scientific literature in the they were able to concede with an expert panel on specific issues studied. Students were however unable to convince other students, who had not participated in the specific activity, that their conclusions were consistent with the scientific evidence and different than what had been taught in class. In many cases, these conclusions differed from their previous perceptions obtained during traditional lectures. In conclusion, the present study suggests that problem-based learning activities should be introduced early in the curriculum to avoid bias in understanding which may occur when students have been previously exposed to information presented in standard lectures. The use of interactive teaching via internet is discussed.

Education, Dental, Graduate↗

PubMed1999

DNA and protein adduct formation in the colon and blood of humans after exposure to a dietary-relevant dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Epidemiology studies have indicated that certain dietary components, including well-cooked meat, are risk determinants for colon cancer. Cooked meat can contain significant quantities of heterocyclic aromatic amines (HCAs), which have been established as carcinogens in laboratory animals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is usually the most mass-abundant HCA, with concentrations up to 480 ppb. We used accelerator mass spectrometry to establish whether DNA and protein adducts can be detected in humans exposed to a quantity of PhIP comparable with levels of exposure that occur in the diet. Five human volunteers were administered a dietary-relevant dose of [14C]PhIP (70-84 microg) 48-72 h before surgery for removal of colon tumors. Blood samples were collected at various time points, and albumin, hemoglobin, and WBC DNA were extracted for analysis by accelerator mass spectrometry. Tissue samples were collected during surgery and used to assess either tissue available doses of [14C]PhIP or adduct levels. The results of this study show: (a) PhIP is activated to a form that will bind to albumin, hemoglobin, and WBC DNA in peripheral blood. WBC DNA adducts were unstable and declined substantially over 24 h; (b) PhIP is bioavailable to the colon, with levels in normal tissue in the range 42-122 pg PhIP/g tissue; and (c) PhIP binds to both protein and DNA in the colon. DNA adduct levels in the normal tissue were 35-135 adducts/10(12) nucleotides, which was significantly lower than tumor tissue. The results of this study demonstrate that PhIP is bioavailable to the human colon following defined dietary-relevant doses and forms DNA and protein adducts.

Adult↗

PubMed1999

Human cytochrome P4501A2.

CYP1A2, a member of the cytochrome P450 superfamily (CYPs), is involved in the metabolic activation of several carcinogens, among them aromatic and heterocyclic amines, nitroaromatic compounds, mycotoxins and estrogens. Several drugs are also metabolized by CYP1A2. Individual differences in CYP1A2 activity may thus influence individual susceptibility to cancer risk and the therapeutic efficacy of some drugs. In humans, CYP1A2 has been detected only in the liver, where it seems to be regulated by at least two mechanisms, one controlling constitutive levels of expression and another regulating inducibility. Wide interindividual differences in CYP1A2 activity have been described. They may be due to factors such as gender, race, genetic polymorphisms, and exposure to inducers. Higher activity has been shown in men than in women. Wide variation across racial/ethnic groups has been reported. Overall, slow and intermediate CYP1A2 metabolizers represent about 50% of Caucasians, while their frequency in Japanese subjects seems to be much lower. No nucleotide differences that could explain the phenotypic variability of the CYP1A2 gene have been found in any exons, exon-intron junctions, or 5'-flanking regions of the gene. However, two genetic variants have been identified which seem to be associated with CYP1A2 inducibility only. Induction of CYP1A2 activity has been reported as a consequence of cigarette smoking, dietary factors, several drugs, chronic hepatitis, and exposure to polybrominated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Several epidemiological studies have been conducted into the relationship between CYP1A2 activity, alone or in combination with other CYPs, and cancer risk. In the absence of a genotypic assay, only the CYP1A2 phenotype can be assessed at present. Many compounds have been tested as in vitro probes to assess CYP1A2 activity in humans. Currently, caffeine has the best potential for use in epidemiological studies: metabolites of caffeine after coffee consumption are measured as an index of CYP1A2 activity. Variable results have been obtained with caffeine-based methods, the use of some caffeine metabolite ratios having given bimodal or trimodal distributions while others have suggested normal or unimodal distributions. Although the epidemiological studies are limited because only phenotyping data are available, there is a suggestion of increased risk of colon cancer and bladder cancer in subjects with rapid CYP1A2 activity. A higher level of 4-aminobiphenyl-haemoglobin adducts has also been found in moderate smokers with rapid CYP1A2 phenotype than in subjects with slow activity.

Biotransformation↗

PubMed1999

Development of an antipeptide antibody that binds to the C-terminal region of human CYP1B1.

An antipeptide antibody was raised against a 14-mer synthetic peptide (CDFRANPNEPA KMN) corresponding to the amino acid sequence from 491 to 504 of human cytochrome P-450 (CYP)1B1. Rabbit-derived antisera demonstrated the ability to induce moderately high antibody titers (>1:10(5)) as judged by enzyme-linked immunosorbent assay. In Western blot analysis, the purified antibody recognized a single protein band (estimated as 56 kDa) in microsomes prepared from human and rodent tissues. No significant cross-reactivity to either human CYP1A1 or human CYP1A2 protein was detected. Titration studies using recombinant human CYP1B1 and an enhanced chemiluminescence-based detection method demonstrated a minimal detection sensitivity for this antiserum at about 0.34 ng/band in 8 x 7-cm minigels. The immunoprecipitation and immunoinhibition results indicate that this antisera recognizes the nondenatured human CYP1B1 protein but does not inhibit its enzyme activity. Using this antibody, CYP1B1 protein was detected in nine different human tissues and in cultured cells induced by various chemicals. This highly specific, highly sensitive antibody provides an important tool to study tissue distribution and cellular expression levels of CYP1B1, with negligible cross-reactivity from the other members of the CYP1 family.

Amino Acid Sequence↗

PubMed1998

Identification of enzymes responsible for the metabolism of heme in human platelets.

The major enzymes involved in the degradation of heme were identified in human platelets. It was determined that heme oxygenase activity levels in umbilical cord blood platelets were higher, whereas biliverdin reductase activity levels were comparable with that found in platelets from adults. In membranes prepared from adenosine diphosphate-activated platelets, UDP-glucuronic acid-dependent bilirubin conjugation was detected, whereas activity was negligible in unactivated platelets and undetected in serum and heat-inactivated platelets, and in platelets prepared from umbilical cord blood. Platelet fractions were analyzed by Western blot and shown to express heme oxygenase, biliverdin reductase, and UDP-glucuronosyltransferases, and there was concordance with known developmental profiles found in other tissues. Heme oxygenase expression was higher, whereas UGT expression was lower, in neonatal compared with adult platelets. These data suggest that platelets are involved in multiple steps of heme and bilirubin metabolism and that developmental regulation of these enzymes may be similar to that in other human tissues.

Adult↗