Polycyclic aromatic hydrocarbon (PAH) metabolizing enzyme activities in human lung, and their inducibility by exposure to naphthalene, phenanthrene, pyrene, chrysene, and benzo(a)pyrene as shown in the rat lung and liver.

In order to survey changes and activities in the polycyclic aromatic hydrocarbon (PAH)-metabolizing enzymes implicated in lung cancer susceptibility studies, we investigated enzyme induction by 2-5-ring-sized 'biomarker' PAHs in rat liver and lung, and the activities in five human lung specimens. Naphthalene, phenanthrene, pyrene, chrysene, and benzo[a]pyrene (BaP) were administered to rats for 3 days (25-128 mg/kg/day) and the responses compared with those of model inducers. PAH treatment increased the CYP1A-catalyzed activity of pyrene 1-hydroxylation and 7-ethoxyresorufin O-deethylation in rat liver by up to 28- and 279-fold, and in rat lung by up to 22- and 51-fold, respectively. 1-Naphthol (hUGT1A6), 1-hydroxypyrene (hUGT1A6/1A9), and entacapone (hUGT1A9) are markers of PAH-glucuronidating human uridine diphosphate-glucuronosyltransferases (UGT). These activities increased up to 6.4-fold in rat liver and up to 1.9-fold in rat lung. NADPH:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase activities increased up to 5.3- and 1.6-fold (liver), and up to 4.4- and 1.4-fold (lung), respectively. CYP1A showed the best liver-to-lung relationship (R (2 )=( )0.90). The inducing efficiency by PAHs differed extensively: control 60-fold), many times greater than the experimental (inducible/constitutive) variation in the rat. Kinetics of 1-hydroxypyrene glucuronidation showed two low-K (m) forms both in rat and human lung. Since the 2-4-ring PAHs (major constituents) were poor enzyme inducers, it appears that the PAH-metabolizing pathways are mainly induced by BaP-type minor constituents. Gene-environmental interactions which magnify polymorphic variability in pulmonary bioactivation/detoxification capacity probably play a key role in individual susceptibility to (or protection against) chemically induced lung cancer. Hence, human exposure to PAH mixtures with high content of BaP-type hydrocarbons confers a potentially higher health risk than PAH mixtures with low content of procarcinogens.

Interactive effects of methyl tertiary-butyl ether (MTBE) and tertiary-amyl methyl ether (TAME), ethanol and some drugs: Triglyceridemia, liver toxicity and induction of CYP (2E1, 2B1) and phase II enzymes in female Wistar rats.

The abilities of the gasoline additives methyl tert-butyl ether (MTBE) and tert-amyl methyl ether (TAME) to cause liver damage following oral administration, dosed alone or in combination with model hepatotoxins, were investigated in the rat. Inducibility of liver drug-metabolizing enzyme activities was also studied. Exposure to these ethers (10-20mmol/kg) for 3 days resulted in hepatomegaly (13-30%) and induction of cytochrome P450 (CYP) activity towards N-nitrosodimethylamine (NDMAD), 7-pentoxyresorufin (PROD), and 7-ethoxyresorufin (EROD). Immunoinhibition assays with monoclonal antibodies showed that the ethers were equipotent as inducers of CYP2E1 activity (2-fold increase) but not of CYP2B1, which was elevated up to 260-fold in TAME-treated rats but only by 20-fold in MTBE rats. A slight or no modifying effect was observed on the NADPH:quinone oxidoreductase (NQO1), glutathione S-transferase (GST), and UDP-glucuronosyltransferase (UGT) activities. Alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) were elevated in blood plasma after administration of the ethers. No dramatic enhancement of liver damage could be detected by plasma enzyme analysis (ALT, AST, alkaline phosphatase, γ-glutamyltransferase) following ether administration (13.5mmol/kg) to rats pretreated with mildly hepatotoxic dosages of ethanol, pyrazole, phenobarbital, acetaminophen (paracetamol), or 13-cis-retinoic acid (13-cis-RA or isotretinoin). Plasma triglycerides increased in TAME-treated rats (1.7-fold) and in all 13-cis-RA-treated groups (2.1-2.8-fold). The findings that MTBE and TAME exhibited a clear but differential inducing effect on two ether-metabolizing CYP forms (2E1 and 2B1) with no marked effect on phase II activities may reflect the importance of these pathways in vivo. The observation that only TAME by itself induced hypertriglyceridemia while acetaminophen- and 13-cis-RA-induced hypertriglyceridemia were aggravated by both ethers, points to differences in their effects on lipid metabolism. TAME was clearly a more potent CNS depressant than MTBE. There was no marked potentiation of drug/chemical-induced acute liver damage either by MTBE or TAME.

Road pavers' occupational exposure to asphalt containing waste plastic and tall oil pitch.

Waste plastic (WP) and tall oil pitch (T), which are organic recycled industrial by-products, have been used as a binder with bitumen in stone mastic asphalt (SMA) and asphalt concrete (AC). We compared the exposure over one workday in 16 road pavers participating in a survey at four paving sites, using mixes of conventional asphalt (SMA, AC) or mixes containing waste material (SMA-WPT, AC-WPT). The concentrations of 11 aldehydes in air were 515 and 902 microg m(-3) at the SMA-WPT and AC-WPT worksites, being 3 and 13 times greater than at the corresponding worksites laying conventional asphalt. Resin acids (2-42 microg m(-3)), which are known sensitizers, were detected only during laying of AC-WPT. The emission levels (microg m(-3)) of total particulates (300-500), bitumen fumes (60-160), bitumen vapour (80-1120), naphthalene (0.59-1.2), phenanthrene (0.21-0.32), pyrene (<0.015-0.20), benzo(a)pyrene (<0.01) and the sum of 16 PAHs (polycyclic aromatic hydrocarbons, 1.28-2.00) were similar for conventional and WPT asphalts. The dermal deposition of 16 PAHs on exposure pads (on workers' wrist) was low in all pavers (0.7-3.5 ng cm(-2)). Eight OH-PAH biomarkers of naphthalene, phenanthrene and pyrene exposures were quantified in pre- and post-shift urine specimens. The post-shift concentrations (mean +/- SD, micromol mol(-1) creatinine) of 1- plus 2-naphthol; 1-,2-,3-,4- plus 9-phenanthrol; and 1-hydroxypyrene were, respectively, for asphalt workers: 18.1+/- 8.0, 2.41 +/- 0.71 and 0.66+/- 0.58 (smokers); 6.0+/- 2.3, 1.70+/- 0.72 and 0.27+/- 0.15 (non-smokers); WPT asphalt workers: 22.0+/- 9.2, 2.82+/- 1.11 and 0.76+/- 0.18 (smokers); 6.8+/- 2.6, 2.35+/- 0.69 and 0.46+/- 0.13 (non-smokers). The work-related uptake of PAHs was low in all pavers, although it was significantly greater in smokers than in non-smokers. The WPT asphalt workers complained of eye irritation and sore throat more than the pavers who had a much lower exposure to aldehydes and resin acids.

Assessment of soil remediation workers' exposure to polycyclic aromatic hydrocarbons (PAH): biomonitoring of naphthols, phenanthrols, and 1-hydroxypyrene in urine.

Urinalysis of multiple polycyclic aromatic hydrocarbons (PAH) biomarkers has been applied to assess the exposure of soil remediation workers on a former creosote wood impregnation site polluted with creosote oil. The uptake of PAHs was measured in preshift, end-of-shift, evening, and next preshift specimens (n=33) of nine volunteers with diverse tasks, using sensitive HPLC-FD methods. The ranges of biomarker concentrations in urine (nmol/l) were: 1-naphthol (14-159), 2-naphthol (9-166), 1- plus 2-naphthol (35-269), 1-hydroxyphenanthrene (OHPhe) (6-56), 2- plus 3-OHPhe (6-70), 4-OHPhe (1-6), 9-OHPhe (1-7), the sum of phenanthrols (15-135), and 1-hydroxypyrene, OHP (2.2-67). Eight of nine workers had OHP levels higher than the Finnish biological limit value for non-occupationally exposed persons (3nmol/l). A linear correlation was observed between 1- and 2-naphthol (r=0.90). The biomarker OHP correlated well in urine both with the major (1-OHPhe, r=0.96; 2- plus 3-OHPhe, r=0.84) and the minor phenanthrene metabolites (4-OHPhe, r=0.77; 9-OHPhe, r=0.68), and with the sum of all phenanthrols (r=0.94), but not so well with the sum of naphthols (r=0.66, p<0.001). The smokers had 2.9-, 2.2-, and 4.8-fold higher average concentrations of naphthols, phenanthrols, and OHP, respectively, than the non-smokers. The PAH biomarker data (concentrations and diurnal excretion profiles) showed significant work-related exposure in both non-smoking and smoking subjects. The average exposure levels were clearly higher than those we have measured for instance in asphalt paving workers. The workers' exposure should be assessed by biological monitoring, because at this type of outdoor work the dermal and pulmonary uptake of PAHs are both likely. Adequate measures for preventing, particularly, dermal absorption are of crucial importance for reducing the workers' risk of exposure to carcinogens on soil remediation sites.

Oxidative stress induced by fumonisin B1 in continuous human and rodent neural cell cultures.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides, which is a common infectant of corn and other cereal grains. Of concern to human health is also a possible airborne exposure to FB1-producing strains of F. verticillioides, which may grow in moisture-damaged buildings. In this study, we have characterized oxidative stress-related parameters induced by FB1 in three different neural cell lines, human SH-SY5Y neuroblastoma, rat C6 glioblastoma and mouse GT1-7 hypothalamic cells. The cells were exposed to graded doses of FB1 between 0.1 and 100 microM for 0-144 h after which the production of reactive oxygen species (ROS), lipid peroxidation, intracellular glutathione (GSH) levels and cell viability were measured. FB1 caused a dose-dependent increase of ROS production in C6 glioblastoma and GT1-7 hypothalamic cells but was without an effect in SH-SY5Y cells. Decreased GSH levels, increased MDA-formation, indicative of lipid peroxidation and necrotic cell death were observed in all cell lines after incubation with FB1. These findings indicate that FB1 induces oxidative stress in human, rat and mouse neural cell cultures.

Fumonisin B1-induced toxicity and oxidative damage in U-118MG glioblastoma cells.

The mycotoxin fumonisin B1 (FB1) is produced by Fusarium verticillioides, which commonly infects corn and other agricultural products. Fusarium species are also a frequent finding in moisture-damaged buildings, causing possible human exposure to FB1. FB1 is neurotoxic and carcinogenic in a number of animal species. In this study, we have investigated the effects of FB1 on human U-118MG glioblastoma cells. The production of reactive oxygen species (ROS), lipid peroxidation, intracellular reduced glutathione (GSH) levels, cell viability, caspase-3-like protease activity and DNA fragmentation were studied in cells exposed to 0.01-100 microM FB1 for 0.5-144 h. FB1 increased lipid peroxidation and the production of ROS in U-118MG cells, showing significant effects after culture times from 48 to 144 h at dose levels of 10 or 100 microM FB1. These effects were accompanied by changes in the GSH levels and cell viability, which decreased significantly after incubating the cells for 48-144 h with the toxin. Signs of apoptosis were indicated by increased caspase-3-like protease activity and internucleosomal DNA fragmentation. Thus, oxidative stress and apoptosis may be involved in the neurotoxicity induced by FB1.

Assessment of catechol induction and glucuronidation in rat liver microsomes.

Catechols are substances with a 1,2-dihydroxybenzene group from natural or synthetic origin. The aim of this study was to determine whether catechols (4-methylcatechol, 4-nitrocatechol, 2,3-dihydroxynaphthalene) and the antiparkinsonian drugs, entacapone and tolcapone, at doses 150 to 300 mg/kg/day, for 3 days, are able to enhance their own glucuronidation. The induction potency of catechols on rat liver UDP-glucuronosyltransferases (UGTs) was compared with that of a standard polychlorinated biphenyl (PCB) inducer, Aroclor 1254. The glucuronidation rate of these catechols was enhanced up to 15-fold in the liver microsomes of PCB-treated rats, whereas treatment with catechols had little effect. Entacapone, tolcapone, 4-methylcatechol, catechol, 2,3-dihydroxynaphthalene, and 4-nitrocatechol were glucuronidated in control microsomes at rates ranging from 0.12 for entacapone to 22.0 nmol/min/mg for 4-nitrocatechol. Using 1-naphthol, entacapone, and 1-hydroxypyrene as substrates, a 5-, 8-, and 16-fold induction was detected in the PCB rats, respectively, whereas the catechol-induced activities were 1.1- to 1.5-fold only. Entacapone was glucuronidated more efficiently by PCB microsomes than by control microsomes (Vmax/Km, 0.0125 and 0.0016 ml/min/mg protein, respectively). Similar kinetic results were obtained for 1-hydroxypyrene. The Eadie-Hofstee plots suggested the contribution of multiple UGTs for the glucuronidation of 1-hydroxypyrene (Km1, Km2, Km3 = 0.8, 9.7, and 63 microM, and Vmax1, Vmax2, Vmax3 = 11, 24, and 55 nmol/min/mg, respectively), whereas only one UGT could be implicated in the glucuronidation of entacapone (Km = 130 microM, Vmax = 1.6 nmol/min/mg). In conclusion, catechols are poor inducers of their own glucuronidation supported by several UGT isoforms. Their administration is unlikely to affect the glucuronidation of other drugs administered concomitantly.

Methylation of cytochrome P4501A1 promoter in the lung is associated with tobacco smoking.

Cytochrome P4501A1 (CYP1A1), which is involved in the metabolic activation of polycyclic aromatic hydrocarbon procarcinogens derived from tobacco smoke, is induced in the lung up to 100-fold because of tobacco smoking. Our aim was to study whether promoter methylation has any role in the smoking-associated expression of CYP1A1 in human lung. Methylation of CpG sites up to 1.4 kb upstream of CYP1A1 gene was studied first by sequencing. Because methylation was observed between nucleotides -1400 and -1000, a methylation-specific single-strand conformational polymorphism method was designed for the region between nucleotides -1411 and -1295 that contains five potential methylation sites, one of them at the xenobiotic responsive element core sequence. Single-strand conformational polymorphism was used on DNA from normal lung samples and peripheral WBCs of smokers and nonsmokers, and on human lung adenocarcinoma (A549) and bronchial epithelial (Beas-2B) cell lines. In lung tissue complete or partial methylation occurred in 33% of heavy smokers (>15 cigarettes/day, n = 30), 71% of light smokers (< or =15 cigarettes/day or quitted 1-7 days earlier, n = 42), and in 98% of nonsmokers (never and ex-smokers, n = 49). Methylation was found to increase in 1-7 days after quitting smoking. In active smokers the lack of methylation in the studied region of CYP1A1 promoter was associated with a slightly higher pulmonary 7-ethoxyresorufin O-deethylase activity in the regression models allowing for the daily tobacco consumption and age. No association was observed in WBC between methylation and tobacco smoking. In lung-derived cell lines the methylation remained stable regardless of induction with benzo(a)pyrene, but a higher induction was observed in Beas-2B cells, which also had less methylation than A549 cells. The association of tobacco smoking with CYP1A1 methylation in the lung suggests that promoter methylation is involved in the regulation of CYP1A1 induction in vivo.

Simultaneous analysis of naphthols, phenanthrols, and 1-hydroxypyrene in urine as biomarkers of polycyclic aromatic hydrocarbon exposure: intraindividual variance in the urinary metabolite excretion profiles caused by intervention with beta-naphthoflavone induction in the rat.

Two fluorimetric HPLC methods are described for the quantification of naphthols, phenanthrols and 1-hydroxypyrene (1-OHP) in urine specimens obtained from male Wistar rats exposed to naphthalene, phenanthrene and pyrene. The polycyclic aromatic hydrocarbons (PAHs) were given intraperitoneally, either alone (1.0 mmol/kg body weight) or as an equimolar mixture (0.33 mmol/kg), using the same dosages for repeated treatments on week 1 and week 2. Between these treatments, PAH-metabolizing activities encoded by aryl hydrocarbon (Ah) receptor-controlled genes were induced in the rats with beta-naphthoflavone (betaNF). Chromatographic separation of five phenanthrols (1-, 2-, 3-, 4-, and 9-isomers) was accomplished using two different RP C-18 columns. Despite selective detection (programmable wavelengths), the quantification limits in the urine ranged widely: 1-OHP (0.18 microg/l)

Enzyme-assisted synthesis and structure characterization of glucuronide conjugates of methyltestosterone (17 alpha-methylandrost-4-en-17 beta-ol-3-one) and nandrolone (estr-4-en-17 beta-ol-3-one) metabolites.

A new and useful method based on enzyme-assisted synthesis was developed for producing 3 alpha-O-beta-D-glucuronide conjugates from synthetic phase I metabolites of methyltestosterone and nandrolone. The formed glucuronide conjugates of 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (I), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (II), 5 alpha-estran-3 alpha-ol-17-one (III), and 5 beta-estran-3 alpha-ol-17-one (IV) are urinary metabolites, indicating the human misuse of the above-mentioned anabolic androgenic steroids (AAS). The common lack of reference material precludes the use and validation of these biomarkers in human doping control. Liver microsomes from Aroclor 1254-induced rats were used as a highly active source of mammalian UDP-glucuronosyltransferases (UGT, EC 2.4.1.17). After purification by protein precipitation, liquid-liquid extraction (dichloromethane), C-18 solid-phase extraction, and lyophilization, the steroid glucuronide structures were characterized by (1)H and (13)C NMR spectroscopy and tandem mass spectrometry. The enzymatic method was highly stereoselective, producing a single major conjugate from the parent steroids I-IV. The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (1.0-2.8 mg, yield 12-29%), which is sufficient for veterinary and human doping control analyses; for pharmaco-, toxico-, and enzyme kinetic studies in the pharmaceutical industry; for clinical laboratories; and for forensic medicine. A new sensitive LC-MS method was developed for controlling the product purity in syntheses, as well as for enzyme kinetic characterization of AAS-metabolizing UGT activities in rat liver toward the aglycones I-IV. In this study, the UGT enzymes responsible for the formation of 3 alpha-O-linked glucuronides from the substrates I, II, III, and IV exhibited the specific enzyme activity values: 25, 124, 48, and 212 nmol/mg microsomal protein in a 2-h incubation, respectively.

Characterization of catechol glucuronidation in rat liver.

Catechols are a class of substances from natural or synthetic origin that contain a 1,2-dihydroxybenzene group. We have characterized the glucuronidation by rat liver microsomes and by the rat liver recombinant UDP-glucuronosyltransferase isoforms UGT1A6 and UGT2B1 of a series of 42 structurally diverse catechols, including neurotransmitters, polyphenols, drugs, and catechol estrogens. Small catechols (4-nitrocatechol, 2,3-dihydroxybenzaldehyde, 4-methylcatechol, and tetrachlorocatechol), tyrphostine A23, and octylgallate were glucuronidated at the highest rate by rat liver microsomes and the recombinant enzymes. By contrast, polyphenols from green tea (catechin and related compounds), 3,5-dinitrocatechol, the catechol-O-methyltransferase inhibitor drugs (entacapone, nitecapone, and tolcapone), the carboxyl catechols (gallic acid and dihydroxybenzoic acid derivatives), and the neurotransmitters and dopaminergic drugs, except dobutamine, were glucuronidated at low rate. Glucuronidation of most catechols was increased upon treatment of rats by 3-methylcholanthrene (3-MC) or Aroclor 1254. No induction was observed after administration of phenobarbital and clofibrate or treatment with catechols. Partial least-squares modeling was carried out to explain the variations of glucuronidation activity by liver microsomes of nontreated and 3-MC-treated rats. The model developed explained 82% and predicted 61% of the variations of glucuronidation activities. Among the 17 electronic and substructure parameters used that characterize the catechols, the hydrophobicity/molar volume ratio of catechols showed a strong positive correlation with the glucuronidation rate. The effect of the pK(a) of the catechol group was modeled to be nonlinear, the optimal pK(a) value for glucuronidation being between 8 and 9. Hydrogen bonding and steric effects also were important to account for to predict the glucuronidation rates.