Multiple endocrine neoplasia type 1 in Northern Finland; clinical features and genotype phenotype correlation.

OBJECTIVE: The existence of genotype-phenotype correlation in multiple endocrine neoplasia type 1 (MEN1) is controversial. Two founder mutations of the MEN1 gene in Northern Finland gave us an opportunity to compare clinical features among heterozygotes of different mutations. DESIGN AND METHODS: Study cohort included 82 MEN1 heterozygotes who were tested for MEN1 during the years 1982-2001. Medical records were reviewed for manifestations of MEN1, other tumours and cause of death by the end of August 2003. Logistic regression analysis was used in evaluating the impact of age, gender and mutational status of affected heterozygotes on the likelihood of developing manifestations of MEN1. RESULTS: Founder mutations 1466del12 and 1657insC were found in 39 and 29 individuals, and D418N, G156R and R527X mutations in 9, 3 and 2 individuals respectively. Except for pituitary adenoma and nonfunctional pancreatic tumour (NFPT), age was a risk factor for all the disease manifestations. For NFPT, frameshift/nonsense mutations (1657insC, R527X) gave an odds ratio (OR) of 3.26 (95% confidence intervals (CI), 1.27-8.33; P = 0.014) compared with in-frame/missense mutations (1466del12, D418N, G156R); including the founder mutation carriers (n = 68) only, the 1657insC mutation gave an OR of 3.56 (CI, 1.29-9.83; P = 0.015). For gastrinoma, in-frame/missense mutations predicted the risk with an OR of 6.77 (CI, 1.31-35.0; P = 0.022), and in the founder mutations group the 1466del12 mutation gave an OR of 15.09 (CI, 1.73-131.9, P = 0.014). CONCLUSIONS: In this study population, NFPT was more common in the frameshift/nonsense or 1657insC mutation carriers, whereas gastrinoma was more common in the in-frame/missense or 1466del12 mutation carriers.

CYP1A1 levels in lung tissue of tobacco smokers and polymorphisms of CYP1A1 and aromatic hydrocarbon receptor.

Induction of a polycyclic aromatic hydrocarbon-metabolizing cytochrome P450 isoform CYP1A1 is regulated by aromatic hydrocarbon receptor (AHR). High inducibility of CYP1A1, possibly due to genetic polymorphisms, has been considered to be a risk factor for lung cancer in tobacco smokers. The relationship between low or high pulmonary expression of CYP1A1 and polymorphic genotypes of CYP1A1 and AHR was investigated in 73 active smokers. CYP1A1 expression was determined in surgical lung samples by measuring ethoxyresorufin O-deethylase (EROD) activity and by immunostaining for CYP1A1 protein. The most common allelic variants of CYP1A1 and AHR in Finns, i.e. the MspI variant (CYP1A1*2A), I462V variant (CYP1A1*2B), and -459C to T variant of CYP1A1 and the R554K variant (AHR*2) of AHR were studied using polymerase chain reaction based methods. EROD activity correlated positively with the daily cigarette consumption (r = 0.45). There was additional variation in EROD activity independent of the amount of smoking e.g. among those who smoked one pack per day until the day of operation, EROD activity ranged from 4-142 (median 48) pmol/min/mg. The frequencies of the MspI, 462V, and -459T variant alleles of CYP1A1 and 554K variant allele of AHR were 0.158, 0.055, 0.055 and 0.075, respectively. No differences were observed in the frequencies of polymorphic genotypes between the smokers with low and those with high expression, when the relationship was studied using a regression analysis adjusted for cigarette consumption. Our results thus indicate that the interindividual variation of CYP1A1 levels in smokers' lung tissue is not attributable to genetic polymorphisms of CYP1A1 or AHR tested in this study.

Glucuronidation of 1-hydroxypyrene by human liver microsomes and human UDP-glucuronosyltransferases UGT1A6, UGT1A7, and UGT1A9: development of a high-sensitivity glucuronidation assay for human tissue.

Human UDP-glucuronosyltransferases (UGT, EC 2.4.1.17) involved in the biotransformation of pyrene were investigated by a sensitive fluorometric high-performance liquid chromatography (HPLC)method developed for determining activities toward 1-hydroxypyrene. The endpoint metabolite of pyrene, 1-pyrenylglucuronide, is a well-known urinary biomarker for the assessment of human exposure to polycyclic aromatic hydrocarbons. 1-Pyrenylglucuronide was synthesized using rat liver microsomes as biocatalyst. The yield was satisfactory, 22%. 1-Pyrenylglucuronide, identified by (1)H NMR and by electrospray mass spectrometry, was used for method validation and calibration. The HPLC assay was very sensitive with a quantitation limit of 3 pg (8 fmol) for 1-pyrenylglucuronide. The assay was precise, showing a relative standard deviation of 5% or less at 0.1 to 300 microM 1-hydroxypyrene. Only 2 microg of microsomal protein was required for the assay in human liver. The glucuronidation of 1-hydroxypyrene was catalyzed at high rates in microsomes from pooled or three individual liver samples, showing comparable apparent K(m) values. The formation of 1-pyrenylglucuronide was catalyzed by recombinant human UGT1A6, UGT1A7, and UGT1A9, the K(m) values being 45, 12, and 1 microM, respectively. The apparent K(m) values in human liver microsomes, ranging from 6.9 to 8.6 microM, agreed well with these results. The method provides a sensitive tool for measuring extremely low UGT activities and a specific means for assessing interindividual differences in 1-hydroxypyrene-metabolizing UGT activities in human liver and other tissues.

Maternal drug abuse and human term placental xenobiotic and steroid metabolizing enzymes in vitro.

We evaluated the impact of maternal drug abuse at term on human placental cytochrome P450 (CYP)-mediated (Phase I) xenobiotic and steroid-metabolizing activities [aromatase, 7-ethoxyresorufin O-deethylase (EROD), 7-ethoxycoumarin O-deethylase (ECOD), pyrene 1-hydroxylase (P1OH), and testosterone hydroxylase], and androstenedione-forming isomerase, NADPH quinone oxidoreductase (Phase II), UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) activities in vitro. Overall, the formation of androstenedione, P1OH, and testosterone hydroxylase was statistically significant between control and drug-abusing subjects; we observed no significant differences in any other of the phase I and II activities. In placentas from drug-abusing mothers, we found significant correlations between ECOD and P1OH activities (p < 0. 001), but not between ECOD and aromatase or P1OH and EROD activities; we also found significant correlations between blood cotinine and UGT activities (p < 0.01). In contrast, in controls (mothers who did not abuse drugs but did smoke cigarettes), the P1OH activity correlated with ECOD, EROD (p < 0.001), and testosterone hydroxylase (p < 0.001) activities. Our results (wider variation in ECOD activity among tissue from drug-abusing mothers and the significant correlation between P1OH and ECOD activities, but not with aromatase or EROD activities) indicate that maternal drug abuse results in an additive effect in enhancing placental xenobiotic metabolizing enzymes when the mother also smokes cigarettes; this may be due to enhancing a "silent" CYP form, or a new placental CYP form may be activated. The change in the steroid metabolism profile in vitro suggests that maternal drug abuse may alter normal hormonal homeostasis during pregnancy.

An uncommon phenotype of poor inducibility of CYP1A1 in human lung is not ascribable to polymorphisms in the AHR, ARNT, or CYP1A1 genes.

Cigarette smoking can induce CYP1A1 in the lung. Induction requires the aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins. Lung samples from seven of 75 Finnish patients who smoked until the time of surgery exhibited absent or low levels of CYP1A1 protein, mRNA and enzymatic activity, suggesting that these individuals might be genetically non or poorly inducible for CYP1A1. All seven lung samples expressed normal levels of AHR mRNA and ARNT mRNA, indicating that they did not carry inactivating polymorphisms in the 5' upstream regulatory regions of these genes. Sequencing of cDNAs encompassing the complete coding regions of AHR and ARNT identified a previously known codon 554 polymorphism in AHR, which was present in the homozygous state in one individual. This polymorphism, which leads to an amino acid substitution, has previously been reported either to have no effect or to enhance CYP1A1 induction. Previously unreported silent single nucleotide polymorphisms were identified in codon 44 of AHR and codon 189 of ARNT. 1500 bp of genomic sequence from the 5' upstream regulatory sequence of the CYP1A1 gene was also sequenced in the non-inducible individuals. A nucleotide substitution polymorphism at position -459 was detected in the heterozygous state in two individuals. This polymorphic site does not reside in any known regulatory sequence. The complete CYP1A1 coding sequence and intron/exon boundaries were then sequenced. None of the non or poorly inducible individuals exhibited any polymorphisms, either homozygous or heterozygous compared to representative inducible individuals or the previously published CYP1A1 sequence. Thus, no polymorphisms in the AHR, ARNT or CYP1A1 genes were identified that could be responsible for the non/low inducibility phenotype observed.

Epoxide hydrolase activity in human blood mononuclear leukocytes: individual differences in native and mitogen-stimulated cells.

Epoxide hydrolase (EH; EC 3.3.2.3) activity was measured in whole-cell sonicates of native and cultured peripheral blood mononuclear cells (PBMCs) from 19 healthy unrelated Caucasian donors (age, 28-55 years). We used 1,2-epoxy-3-(p-nitrophenoxy)propane (0. 34 mM) as the substrate and, for the diol assay, a quantitative HPLC method with spectrophotometric detection. One portion of the PBMCs was frozen immediately, while the other portion was PHA-stimulated and cultivated for 36 h. In native leukocytes, the EH activity varied from 2.2 to 8.2 pmol/min per 10(6) cells (3.8-fold), the mean+/-SD was 5.6+/-1.4 pmol/min per 10(6) cells. In most of the samples from different donors, the specific activity increased in cultivation, varying from 2.4 to 15.4 pmol/min per 10(6) cells (6. 3-fold), the mean+/-SD being 8.5+/-3.8 pmol/min per 10(6) cells. From a methodological point of view, enzyme measurement in native cells is simple to perform and may provide a better index of the specific activity, as the accuracy of electronic cell counting is better for cell samples taken before than after cultivation. The differences in the EH activity of PBMCs indicate that significant interindividual variation may occur in the detoxification of epoxides produced in the human lymphocyte test systems commonly used for genotoxicity screening of chemicals in vitro. Further studies are needed to determine the extent to which the reproducibility and thus also the sensitivity of such assays could be improved by analyses carried out to control the donors for their EH phenotype.

Enzyme-assisted synthesis and structural characterization of nitrocatechol glucuronides.

Enzyme-assisted synthesis and characterization are described for 3-O-beta-D-glucuronides 1b-4b of the aglycons E- and Z-2-cyano-N, N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)propenamide (entacapone), 1a and 2a, respectively, 3-(3,4-dihydroxy-5-nitrobenzylidene)-2, 4-pentanedione (nitecapone) 3a and 4'-methyl-3, 4-dihydroxy-5-nitrobenzophenone (tolcapone) 4a, and 1-o- and 2-o-glucuronides 5b and 6b of the aglycon 1, 2-dihydroxy-4-nitrobenzene 5a. Liver microsomes from rats pretreated with Aroclor 1254 were used as catalyst in the synthesis. Glucuronidation was regio- and stereoselective in the case of 1a-4a; only one product was observed by HPLC, HPTLC, and NMR. The glucuronidation of 1,2-dihydroxy-4-nitrobenzene 5a resulted in equal amounts of 1-O-beta-D- and 2-O-beta-D-glucuronides. Purification of the crude products by C18 solid-phase extraction and/or flash chromatography gave compounds 1b-6b in 38-98% yields (50-84 mg). The structures of the glucuronides were characterized on the basis of UV and IR spectra and confirmed with FAB-MS and NMR spectroscopy.

Glucuronidation of entacapone, nitecapone, tolcapone, and some other nitrocatechols by rat liver microsomes.

PURPOSE: Nitrocatechol COMT inhibitors are a new class of bioactive compounds, for which glucuronidation is the most important metabolic pathway. The objective was to characterize the enzyme kinetics of nitrocatechol glucuronidation to improve the understanding and predicting of the pharmacokinetic behavior of this class of compounds. METHODS: The glucuronidation kinetics of seven nitrocatechols and 4-nitrophenol, the reference substrate for phenol UDP-glucuronosyltransferase activity, was measured in liver microsomes from creosote-treated rats and determined by non-linear fitting of the experimental data to the Michaelis-Menten equation. A new method that combined densitometric and radioactivity measurement of the glucuronides separated by HPTLC was developed for the quantification. RESULTS: Apparent K(m) values for the nitrocatechols varied greatly depending on substitution pattern being comparable with 4-nitrophenol (0.11 mM) only in the case of 4-nitrocatechol (0.19 mM). Simple nitrocatechols showed two-fold Vmax values compared with 4-nitrophenol (68.6 nmol min-1 mg-1), while all disubstituted catechols exhibited much lower glucuronidation rate. Vmax/K(m) values were about 10 times higher for monosubstituted catechols compared to disubstituted ones. The kinetic parameters for COMT inhibitors were in the following order: K(m) nitecapone > > entacapone > tolcapone; Vmax nitecapone > entacapone > tolcapone; Vmax/K(m) tolcapone > nitecapone > entacapone. CONCLUSIONS: Nitrocatechols can in principle be good substrates of UGTs. However, substituents may have a remarkable effect on the enzyme kinetic parameters. The different behaviour of nitecapone compared to the other COMT inhibitors may be due to its hydrophilic 5-substituent. The longer elimination half-life of tolcapone in vivo compared to entacapone could not be explained by glucuronidation kinetics in vitro.

Cytochrome P450 isozymes responsible for the metabolism of toluene and styrene in human liver microsomes.

1. Cytochrome P450 isozymes from Asian (31 Chinese subjects) and Caucasian (14 Finnish subjects) livers were examined for their roles in the metabolism of toluene (rates of benzyl alcohol, o- and p-cresol formation) and styrene (rates of styrene glycol formation). 2. For toluene, the overall rate of metabolism was higher in samples from Finnish than from Chinese subjects. At 0-20 mM toluene, the rate of o-cresol formation was significantly higher in Finnish microsomes than in Chinese ones. The formation rates of benzyl alcohol and p-cresol in Finnish samples were also higher than those of Chinese samples, but only at a high substrate concentration (5.0 mM). For styrene metabolism, the Chinese liver microsomes showed higher metabolic rates than the Finnish ones at 0.085 mM styrene, but not at the higher substrate concentration. 3. Mean expression levels of immunochemically detected CYP1A2/1 and CYP2B6 were almost 3-fold higher in Finnish microsomes, whereas CYP2E1 was 1.7-fold higher in Chinese samples. 4. Correlation analysis showed that CYP2E1 (benzyl alcohol formation) and CYP1A2/1 (o-cresol formation) contributed to the metabolism of toluene at the low substrate concentration, whereas CYP2C8 was the form more actively involved at the higher toluene concentrations. At the higher concentration (1.8 mM) of styrene, CYP2B6 was most active isozyme to catalyse the formation of styrene oxide from styrene. 5. These results suggest that CYP2E1 and CYP1A2/1 are the main isoforms responsible for the metabolism of toluene at low substrate concentrations in human liver microsomes, CYP2E1 at low styrene concentration, and CYP2C8 and CYP2B6 at high concentrations of toluene and styrene respectively.

Characterization of 1-hydroxypyrene as a novel marker substrate of 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase(s).

Rats were treated with acetone, pyrazole, phenobarbital, 4,4'-methylenebis-(2-chloroaniline) (MOCA), 3-methylcholanthrene, creosote oil, or a mixture of polychlorinated biphenyls (Aroclor 1254) to study the inducibility and enzyme kinetics of UDP-glucuronosyltransferases towards 1-hydroxypyrene, which is a human metabolite and a urinary biomarker of exposure to pyrene. The rate of 1-hydroxypyrene glucuronidation was analyzed in rat liver microsomes by a fluorometric HPLC assay of the formed glucuronide. The apparent K(m) and Vmax values in untreated controls (K(m) = 0.27 mM; Vmax = 31 nmol/min./mg protein) did not differ markedly from those in rats treated with acetone, pyrazole or phenobarbital, whereas the significantly decreased K(m) and increased Vmax values of the rats treated with the carcinogenic chemicals, MOCA (0.11; 51), creosote (0.06; 137), 3-methylcholanthrene (0.07; 141) or the Aroclor-1254 polychlorinated biphenyl (PCB) mixture (0.08; 226), implicated major changes in the hepatic expression of UDP-glucuronosyltransferases. 1-Hydroxypyrene proved to be a high affinity substrate and a sensitive marker of the polycyclic aromatic hydrocarbon (PAH) metabolizing UDP-glucuronosyltransferase(s). Catalytically, the most efficient isoforms were induced in creosote, 3-methylcholanthrene and PCB-treated rats showing Vmax/K(m) ratios which were 22-27 times greater than in untreated controls. Our findings suggest the existence of a 3-methylcholanthrene type inducible and a functionally efficient low-K(m)/ high-Vmax form(s) of UDP-glucuronosyltransferase(s) that detoxify 1-hydroxypyrene and probably other polycyclic aromatic hydrocarbons as well.

Toluene metabolism by cDNA-expressed human hepatic cytochrome P450.

The metabolism of toluene in human liver microsomes and by cDNA-expressed human cytochrome P450s (CYPs) was investigated. Toluene was metabolized mainly to benzyl alcohol and slightly to o- and p-cresol by human liver microsomes. Formation of o-cresol was elevated in microsomes from human livers derived from cigarette smokers, but the induced CYP isoforms were not clear. Of the eleven human CYP forms studied, CYP2E1 was the most active in forming benzyl alcohol, followed by CYP2B6, CYP2C8, CYP1A2, and CYP1A1, in that order. The activities of CYP2A6, CYP2C9, CYP2D6, CYP3A3, CYP3A4, and CYP3A5 were negligible. In addition, CYP2B6 and CYP2E1 catalyzed the formation of p-cresol (11-12% of total metabolites), and CYP1A2 catalyzed the formation of both o-(22%) and p-cresol (35%). The relationship between the amino acid sequence of rat CYP2B1 cDNA and the activity for toluene metabolism was investigated using variants, because of great differences in the forming of toluene ring products between CYP2B1 and CYP2B6. These results suggest that the structure of CYP2B1 at the site of Leu 58 rather than Ile-114 and Glu-282 plays an important role in the formation of toluene ring products, whereas in CYP2B1 Ile-114 plays an important role in the formation of benzyl alcohol. These results may explain, in part, the lower activity of CYP2B6, which has Phe at position 58 of the protein, for toluene ring oxidations than that of CYP2B1.

Different contributions of cytochrome P450 2E1 and P450 2B1/2 to chloroform hepatotoxicity in rat.

The contribution of cytochrome P450 isozymes CYP2E1 and CYP2B1/2 to chloroform-induced hepatotoxicity taken at 18 hr after the treatment was investigated in rats treated with n-hexane as an inducer of CYP2E1, 2-hexanone as an inducer of CYP2E1 and CYP2B1/2, and phenobarbital (PB) as an inducer of CYP2B1/2. Hepatic damage was evaluated by gross measurement of plasma alanine aminotransferase activity and histopathological examination. All treatments potentiated chloroform-induced hepatic damage. In n-hexane-pretreated rats, the damage was maximal with the middle dose of chloroform (0.2 ml/kg), whereas the damage increased with dose in rats treated with 2-hexanone or PB. The degree of hepatic damage induced with the three pretreatments was in the following order: n-hexane > 2-hexanone = PB with the middle dose of chloroform and PB >> 2-hexanone > n-hexane with the high dose (0.5 ml/kg); little difference among the pretreatments was seen with the low dose (0.1 ml/kg). These findings suggest that CYP2E1 is a low Km isoform and CYP2B1/2 a high Km isoform for chloroform activation. CYP2E1-dependent hepatic damage was characterized by ballooned hepatocytes, which were restricted to the centrilobular area; with CYP2B1/2, more necrotic than ballooned hepatocytes were seen and the necrotic hepatocytes were found not only in the centrilobular but also in the midzonal and periportal areas. Chloroform treatment did not affect the activity of N-nitrosodimethylamine N-demethylase in pretreated rats; the high dose increased the activity in control rats. In contrast, the high dose of chloroform decreased the activity of 7-pentoxyresorufin O-depentylase in all induced rats but not in controls. Immunoinhibition and immunoblot analyses showed that the high dose of chloroform induced CYP2E1 in control rats but decreased CYP2B1/2 in all pretreated rats. These results suggest that although both CYP2E1 and CYP2B1/2 contribute to chloroform-induced hepatic damage, they do so quite differently.

Pulmonary expression of glutathione S-transferase M3 in lung cancer patients: association with GSTM1 polymorphism, smoking, and asbestos exposure.

To characterize the relative roles of glutathione S-transferases (GST) M1 and M3 in the susceptibility to lung cancer, the pulmonary expression of GSTM3 was quantified immunochemically and related to the GSTM1 genotype in 100 lung cancer patients. Among active smokers and recent ex-smokers (for 6 years or less), parenchymal GSTM3 expression was lower in patients with a homozygous GSTM1 null genotype than in those who were GSTM1 positive and had similar smoking habits (P < 0.001 and P = 0.004, respectively). However, in long-term ex-smokers (for 15 years or longer) GSTM3 was not affected by the GSTM1 genotype. Among active smokers and recent ex-smokers who were homozygous GSTM1 null, those with a definite or probable exposure to asbestos expressed GSTM3 at significantly higher levels than those for whom it was unlikely (P = 0.04). A similar effect of the homozygous GSTM1 null genotype on GSTM3 expression was not detected in the bronchial epithelium when GSTM3 was visualized immunohistochemically. Different mechanisms may result in an increased risk of either squamous cell or adenocarcinomas in patients with the homozygous GSTM1 null genotype. Low expression of GSTM3 due to smoking in the parenchymal lung of GSTM1 null individuals can theoretically favor the development of adenocarcinoma. Our data indicated a predominance of this tumor type in patients with low expression of GSTM3.

Expression and polymorphism of glutathione S-transferase in human lungs: risk factors in smoking-related lung cancer.

The relationships between smoking and the expression of glutathione S-transferase (GST*) isozymes GSTM1-1, GSTM3-3, GSTP1-1 and GSTA1-1/2-2 (GSTA1/2), or between smoking and activities of epoxide hydrolase (EH) and aryl hydrocarbon hydroxylase (AHH) were investigated in lung samples from 27 patients with lung cancer and 11 control patients by immunoblot analysis and enzyme assays. Determination of genotypes in blood leucocyte DNA showed that possession of the mu-class GSTM1 gene was closely related to the expression of GSTM1-1 and GSTM3-3 enzymes in lung cytosol: patients with the GSTM1 null genotype had no detectable GSTM1 protein and less GSTM3 protein than patients with the GSTM1 gene (P < 0.001). Absence of the GSTM1 gene did not affect the content of phi-class GSTP1-1 or alpha-class GSTA1/2. GST activity towards 1-chloro-2,4-dinitrobenzene was lower (P < 0.01) in patients lacking the GSTM1 gene than in those expressing GSTM1; in general, patients with a low GSTM3-3, GSTP1-1 or GSTA1/2 content also had significantly less overall GST activity. The pulmonary content of GSTP1-1 was greater in cancer than in non-cancer patients (P < 0.05). Smoking did not influence the levels of GST isozymes or the EH activity. In contrast, the AHH activity was significantly (P < 0.01) increased by smoking. Neither AHH nor EH showed a correlation with GSTM1 polymorphism. Our data support the idea that in smokers who lack the GSTM1 gene, activation of carcinogens in tobacco smoke (e.g. benzo[alpha]pyrene) is increased, while the efficacy of detoxification is limited both qualitatively (absence of GSTM1-1 enzyme and low expression of GSTM3-3 enzyme) and quantitatively (low overall GST activity). This imbalance in the metabolism of carcinogens may explain the increased susceptibility to lung cancer reported in smokers with the GSTM1 null genotype.

Significance of dermal and respiratory uptake in creosote workers: exposure to polycyclic aromatic hydrocarbons and urinary excretion of 1-hydroxypyrene.

OBJECTIVES: To evaluate workers' exposure in a creosote impregnation plant by means of ambient and biological monitoring. METHODS: Naphthalene (vapour phase) and 10 large molecular polycyclic aromatic hydrocarbons (PAHs) (particulate phase) were measured in the breathing zone air during an entire working week. 1-Hydroxypyrene (1-HP) was measured in 24 hour urine as a metabolite of the pyrene found in neat (dermal exposure) and airborne creosote. RESULTS: Naphthalene (0.4-4.2 mg/m3) showed 1000 times higher concentrations in air than did the particulate PAHs. In total, the geometric mean (range) of three to six ring PAHs was 4.8 (1.2-13.7) micrograms/m3; pyrene 0.86 (0.23-2.1) micrograms/m3, and benzo(a)pyrene 0.012 (0.01-0.05) micrograms/m3. There was no correlation between pyrene and gaseous naphthalene. The correlations between pyrene and the other nine particulate PAHs were strong, and gave a PAH profile that was similar in all air samples: r = 0.83 (three to six ring PAHs); r = 0.81 (three ring PAHs); r = 0.78 (four to six ring PAHs). Dermal exposure was probably very high in all workers, because the daily output of urinary 1-HP exceeded the daily uptake of inhaled pyrene by < or = 50-fold. Urinary 1-HP concentrations were very high, even on Monday mornings, when they were at their lowest (4-22 mumol/mol creatinine). 1-HP seldom showed any net increase over a workshift (except on Monday) due to its high concentrations (16 to 120 mumol/mol creatinine) in the morning samples. 1-HP was always lower at the end of the shift (19 to 85 mumol/mol creatinine) than in the evening (27 to 122), and the mean (SD) change over the working week (47 (18)) was greater than the change over Monday (35 (32)). The timing of 1-HP sampling is therefore very important. CONCLUSIONS: Urinary 1-HP proved to be a good biomarker of exposure to three to six ring PAHs but not to airborne naphthalene. Hence, biomonitoring based on 1-HP has to be completed with exposure assessment for naphthalene as a marker for creosote volatiles that mainly enter the body through the lungs.

Effect of cytochrome P450 isozyme induction and glutathione depletion on the metabolism of CS2 to TTCA in rats.

Analysis of 2-thiothiazolidine-4-carboxylic acid (TTCA), a metabolite of carbon disulfide (CS2), is used in the biological monitoring exposure to CS2 at work. In order to clarify the metabolic reasons for individual variation in the urinary excretion of TTCA, the latter was studied in rats pretreated with model cytochrome P450 (CYP) enzyme inducers or glutathione (GSH) depletors. Ethanol, phenobarbital (PB) or 3-methylcholanthrene (MC) did not increase 24-h TTCA output following CS2 inhalation (50 or 500 ppm, 6h). After oral dosing (10 mg/rat), PB had an inhibiting effect on the excretion rate of TTCA. Tissue GSH depletors phorone, L-buthionine-(RS)-sulfoximine (BSO) and diethylmaleate (DEM) decreased TTCA excretion in rats given an oral dose (10 mg/rat) of CS2. The initial inhibition by phorone and DEM was reversed after 6 h and from 12 h onward the TTCA in urine exceeded the control level, an effect not seen with BSO. The proportion of CS2 excreted in urine as TTCA within 24 h was 1.7% in control rats and 1% after BSO treatment, 1.3% after PB, 1.7% after acetone, 1.8% after MC, 2.0% after phorone and 2.5% after DEM treatment. The amount of TTCA in urine increased with the CS2 dose in a non-linear fashion: 1.6 mumol (50 ppm/6 h) vs. 4.9 mumol (500 ppm/6 h), and 0.2 mumol (1 mg/kg) versus 3.6 mumol (100 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Styrene metabolism by cDNA-expressed human hepatic and pulmonary cytochromes P450.

The rate of formation of styrene glycol from styrene was compared in human, rat, and mouse liver microsomes. At a low styrene concentration (0.085 mM), the rates decreased in the order, mouse (2.43 +/- 0.29 nmol/(mg of protein.min)) > rat (1.07 +/- 0.20) > human (0.73 +/- 0.45); at a high concentration (1.85 mM), the order was rat (4.21 +/- 0.72) > mouse (2.72 +/- 0.11) > human (1.91 +/- 0.84). Kinetic analysis indicated the presence of at least two forms of styrene-metabolizing cytochrome P450s with different Km values in human liver microsomes. Styrene was also metabolized in human lung microsomes: the rate of styrene glycol formation was higher in the lung microsomes from smokers than in those from current nonsmokers. The P450 forms responsible for transforming styrene to styrene glycol were determined by analyzing cDNA-expressed individual P450 forms produced in cultured hepatoma G2 cells by recombinant vaccinia viruses. Of the 12 human P450 forms studied, CYP2B6 and CYP2E1 existing in human liver and/or lungs and CYP2F1 in human lungs were the most active in the forming of styrene glycol, followed by CYP1A2 and CYP2C8. Human CYP3A3, CYP3A4, CYP3A5, and CYP4B1 also catalyzed the metabolism but were much less active. CYP2A6, CYP2C9, and CYP2D6 had only a little detectable activity. CYP1A2, CYP2B6, CYP2C8, CYP2E1, and CYP3A4/3A3 were expressed in human liver microsomes, and CYP2C8 was expressed in human lung microsomes, although the expression of CYP2F1 and CYP4B1 could not be investigated. These data indicate that several human hepatic and/or pulmonary P450 forms are capable of metabolizing styrene, albeit at different rates.

CYP2C11 and CYP2B1 are major cytochrome P450 forms involved in styrene oxidation in liver and lung microsomes from untreated rats, respectively.

The contribution of cytochrome P450s (P450s) to the formation of styrene glycol from styrene in rat liver microsomes was investigated using monoclonal antibodies to P450s. Anti-CYP2E1 inhibited the formation to a similar extent in ethanol-treated microsomes and in control microsomes in terms of percentage inhibition, whereas to a greater extent in the former than the latter in terms of net inhibition, and only at low substrate concentration. Anti-CYP2C11/6 also inhibited the formation in control and in ethanol-treated microsomes at both low and high concentrations of styrene, and the net degree of inhibition was greater than that obtained with anti-CYP2E1, even in ethanol-treated microsomes where CYP2E1 was induced. Anti-CYP2B1/2 and anti-CYP1A1/2 inhibited the formation only in phenobarbital (PB)- and 3-methylcholanthrene (MC)-induced microsomes, respectively. These results suggest that (1) at least four P450s, CYP2C11/6, CYP2E1, CYP2B1/2 and CYP1A1/2, contribute to the metabolism of styrene, (2) CYP2C11/6, which probably corresponds to CYP2C11, is the major form of P450 responsible for the metabolism in untreated rat liver microsomes, and also in those treated with ethanol. Anti-CYP2E1 inhibited styrene oxidation more prominently in microsomes from styrene-treated rats than in those from control rats at a low substrate concentration. Although styrene treatment did not influence the total metabolism of styrene in liver microsomes at a high substrate concentration, inhibition of the metabolism by anti-CYP2C11/6 decreased with increasing styrene dose, whereas that by anti-CYP2B1/2 increased, suggesting that styrene treatment increases CYP2B1/2 but decreases CYP2C11/6 in rat liver, and the major form of P450 which mediates styrene oxidation is CYP2B1/2 after the treatment. Only anti-CYP2B1/2, which probably corresponds to CYP2B1, inhibited styrene oxidation in lung microsomes from untreated and even styrene-treated rats. Thus, the major form of P450 responsible for the metabolism of styrene is different in each tissue.