Ethoxyresorufin Deethylase Activity Research Articles

Expression and inducibility of CYP1A1, 1A2, 1B1 by β-naphthoflavone and CYP2B22, 3A22, 3A29, 3A46 by rifampicin in the respiratory and olfactory mucosa of pig

The presence and inducibility of specific CYPs (1A1, 1A2, 1B1, 2B22, 3A22, 3A29 and 3A46) and the related transcriptional factors (AhR, CAR, PXR, and HNF4α) were investigated, at activity and/or transcriptional level, in liver, respiratory and olfactory mucosa of control and β-naphthoflavone (βNF)-treated pigs an agonist of AhR, or rifampicin (RIF), an agonist of PXR. Experiments with real-time PCR showed that CYP1A1 mRNA was enhanced by βNF, although at different extent, in liver, respiratory and olfactory tissues, whereas mRNAs of CYP1A2 and 1B1 were increased only in liver. Accordingly, in microsomes of both nasal tissues, the transcriptional activation of CYP1A1 was accompanied by an induction of ethoxyresorufin deethylase activity (a marker of this isoform) but not of methoxyresorufin demethylase activity (a marker of CYP1A2). The rifampicin treatment resulted in a transcriptional activation of CYP2B22 and CYP3As genes in liver but not in respiratory and olfactory mucosa. In parallel, the marker activity of CYP2B (ethoxy 4-(trifluoromethyl)coumarin deethylase) and CYP3As (6β-testosterone hydroxylase and benzyloxyquinoline debenzylase) were induced in liver microsomes but not in the nasal ones. Considering the transcriptional factors, the basal expression of AhR mRNA was found to be as high in liver as in both nasal tissues but not susceptible to induction by βNF. Also PXR mRNA was found, aside liver, well expressed in the nasal tissues, whereas CAR and HNF4α mRNAs were barely detected. In any case, these transcripts appeared to be enhanced by RIF treatment. Our results demonstrated that in the respiratory and olfactory mucosa of pig, although the presence of AhR, only CYP1A1, but not 1A2 and 1B1 resulted to be inducible by βNF. Similarly, it was observed that in these nasal tissues, although the presence of PXR, neither CYP2B22 nor any CYP3A resulted to be inducible by RIF. Thus, the regulation mechanism of CYP1A2, 1B1, 2B22, 3A22, 3A29, and 3A46, in the nasal mucosa involves tissue-enriched transcriptional factors others than AhR, CAR, PXR, and HNF4α, which are fundamental in liver.

Bone biomechanical property deterioration due to tobacco smoke exposure.

Tobacco smoking has been implicated in the development of osteoporosis and early onset of menopause in women smokers. We measured various biomechanical properties of femurs and tibiae obtained from smoke-exposed and control mice to determine cigarette smoke influences on bone mass, structure, and strength. Growing female C57BL mice were exposed to sidestream cigarette smoke in a whole-body exposure chamber, set at 30 +/- 2 mg smoke particulates/m3 for 4 hours/day and 5 days/week for 12 consecutive weeks. Elevated levels of urinary cotinine and pulmonary ethoxyresorufin deethylase activity in smoke-exposed mice confirmed their effective exposure to cigarette smoke. There were no differences in body weight and physical size (length, medial-lateral and anterior-posterior widths, midshaft cortical area and thickness) of femurs and tibiae between smoke-exposed and control mice. The femoral mid-shaft yield load, stiffness, yield stress, and modulus were, respectively 8%, 13%, 10%, and 14% lower (P < 0.05) in smoke-exposed compared to control mice. The ultimate load and stress in mid-shaft femurs showed decreasing trends (P < 0.1) in smoke-exposed mice. In the femoral neck, the ultimate load and stiffness were 9% and 12% lower (P < 0.05) in smoke-exposed mice, respectively. Further, the ash-to-dry bone weight ratio was smaller ( approximately 6%, P < 0.05), and micro-computed tomographic scanning of distal femoral bone volume/total volume (%) and trabecular thickness showed decreasing trends in smoke-exposed mice compared to the control group. We conclude that exposure to tobacco smoke deteriorates some of the biomechanical properties of bone in growing female mice.

Subchronic toxicity of Baltic herring oil and its fractions in the rat II: Clinical observations and toxicological parameters.

This study aimed to increase the knowledge about the toxicity of fish-derived organohalogen pollutants in mammals. The strategy chosen was to separate organohalogen pollutants derived from Baltic herring (Clupea harengus) fillet, in order to obtain fractions with differing proportions of identified and unidentified halogenated pollutants, and to perform a subchronic toxicity study in rats, essentially according to the OECD guidelines, at three dose levels. Nordic Sea lodda (Mallotus villosus) oil, with low levels of persistent organohalogen pollutants, was used as an additional control diet. The toxicological examination showed that exposure to Baltic herring oil and its fractions at dose levels corresponding to a human intake in the range of 1.6 to 34.4 kg Baltic herring per week resulted in minimal effects. The spectrum of effects was similar to that, which is observed after low-level exposure to pollutants such as chlorinated dibenzo-p-dioxins and dibenzofurans (CDD/F) and chlorinated biphenyls, despite the fact that these contaminants contribute to a minor part of the extractable organically bound chlorine (EOCI). The study confirmed previous findings that induction of hepatic ethoxyresorufin deethylase (EROD) activity takes place at daily intake levels 0.15 ng fish-derived CDD/F-TEQs/kg body weight. The study also demonstrated that hepatic vitamin A reduction takes place at somewhat higher daily exposure levels, i.e. 0.16-0.30 ng fish-derived CDD/F-TEQs/kg body weight. Halogenated fatty acids, the major component of EOCI, could not be linked to any of the measured effects. From a risk management point of view, the study provides important new information of effect levels for Ah-receptor mediated responses following low level exposure to organohalogen compounds from a matrix relevant for human exposure.

Human hepatocyte cell lines proliferating as cohesive spheroid colonies in alginate markedly upregulate both synthetic and detoxificatory liver function

Background/Aims: Bio-artificial liver support systems for treatment of hepatic failure require maintained expression of hepatocyte function in vitro. We studied cultures of human hepatocyte cell-lines proliferating within alginate beads, investigating the hypothesis that 3-dimensional cohesive colonies of hepatocyte cell-lines would achieve polarity and cell-to-cell contact resulting in upregulation of function. Methods: HepG2 and HHY41 human cell lines in alginate beads were cultured for >20 days. Results: Proliferation was maintained for 20 days. Production of albumin, prothrombin, fibrinogen, alpha-1-acid glycoprotein and alpha-1-antitrypsin was maintained throughout, maximal at days 8–10, when upregulation was 300–1100% compared with monolayer cultures at similar cell number per unit volume. Detoxificatory functions: ethoxyresorufin deethylase activity, androstenedione metabolism, and urea synthesis from arginine was also increased several-fold. Function returned to pre-freezing levels within 18 h of thawing after cryopreservation of cells in alginate. Electron microscopy revealed spherical colonies of cells of cuboidal shape, with cell-to-cell contact via desmosomes and junctional complexes, abundant microvilli, and cytoplasmic appearances suggesting transcriptionally active hepatocytes. Conclusion : Hepatocyte cell-lines, proliferating in alginate express a range of liver-specific functions at levels approaching those found in vivo, relevant to their use in liver support systems.

Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/ insect cell system

1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (I462V) and CYP1A1.4 (T461N), were co-expressed with human NADPHP450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus coinfection to elaborate a suitable system for studying the role of CYP1A1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. Under optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethylase activities (nmol/min-1 nmol-1 CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated ~ 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B\\[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-coinfection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CYP1A1 variants.

Elevated levels of cytochrome P4501A (CYP1A) in ringed seals from the Baltic Sea

The high frequency of a variety of disorders observed in Baltic seals have been correlated with elevated concentrations of organochlorines. For this study, we determined hepatic CYP1A activities and apoprotein levels in ringed seals ( Phoca hispida) exposed to high levels of organochlorides in the Baltic Sea and in corresponding seals from a relatively unpolluted area (Svalbard Region). Activities of ethoxyresorufin deethylase (EROD) and pentoxyresorufin depentylase (PROD) in seal liver microsomes were increased about 3-fold in the Baltic sample, the EROD activity reaching similar levels to those found in 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced rats (about 1,2 nmol/mg protein per min). No sex-related differences in EROD or PROD activities were observed in either population. Hepatic CYP1A levels in ringed seals were further characterised by inhibition and Western blot analysis. α-Naphthoflavone and furafylline were used as chemical inhibitors, having highly specific inhibiting effects on CYP1A1 and CYP1A2, respectively, in rat and human liver. α-Naphthoflavone inhibited potently both EROD and PROD activities, while inhibition by furafylline was weaker. A monoclonal antibody specific to CYP1A in the rat (MAb 1-7-1), used in the immunoblots, revealed a substantially increased amount of CYP1A protein in the Baltic seals. CYP2B-related proteins were not detected in any seal sample using MAb 2-66-3, which is specific for rat CYP2B. Thus, both inhibition and Western blotting experiments indicate that CYP1A is mainly responsible for the marked increase in microsomal EROD and PROD activities in the livers of Baltic ringed seals. Since the properties of all CYP enzymes vary between species, further studies are needed to verify whether CYP1A1 or CYP1A2 mediates the observed EROD induction in the ringed seal.

Differences in kinetics of induction and reversibility of TCDD-induced changes in cell proliferation and CYP1A1 expression in female Sprague-Dawley rat liver.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent tumor promoter in two-stage initiation-promotion models and induces cell proliferation and development of enzyme-altered hepatic foci. It is believed that increased cell proliferation is a necessary step in carcinogenesis. Therefore, the analysis of the effect of TCDD on cell proliferation in rat liver may aid in the understanding of the mechanism of hepatocarcinogenesis induced by TCDD. The aim of this study was to investigate the time course and reversibility of cell proliferation in non-initiated and diethylnitrosamine-initiated female rats exposed biweekly to a daily averaged dose of 125 ng TCDD/kg/day for up to 60 weeks. In addition we evaluated the suitability of different dose metrics for the evaluation of TCDD-induced changes in cell proliferation and CYP1A1 enzyme induction. Cell proliferation was measured as the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into hepatocytes undergoing replicative DNA synthesis. Mean BrdU labeling indices in TCDD-treated animals were not increased over controls after 14 weeks exposure, but were increased 8- and 2-fold after 30 and 60 weeks' treatment respectively, despite similar liver levels of TCDD at all these times (23-30 p.p.b.). In comparison, CYP1A1 activity, as measured by ethoxyresorufin deethylase activity, was significantly induced at all times points analyzed. Sixteen weeks following cessation of TCDD treatment, labeling indices were still significantly elevated over controls, but after 30 weeks of withdrawal, labeling indices were no different from controls, indicating that TCDD-induced changes in cell proliferation were reversible. Dosimetric analysis indicated that rat liver tissue burden was suitable for prediction of CYP1A1 expression but not cell proliferation and that the area under the curve was unsuitable for prediction of both TCDD-induced changes in CYP1A1 expression and cell proliferation.

Subchronic toxicity of PCB 105 (2,3,3',4,4'-pentachlorobiphenyl) in rats.

The toxicity of 2,3,3',4,4'-pentachlorobiphenyl (PCB 105) was investigated in Sprague-Dawley rats following dietary exposure to this substance at levels of 0, 0.05, 0.5, 5 or 50 ppm for 13 weeks. Growth rate and food consumption were not affected and no clinical signs of toxicity were observed. Increased incidences of enlarged, fatty liver and decreased thymic weight were observed in the highest-dose groups of both genders; these groups also had elevated hepatic microsomal ethoxyresorufin deethylase activity and uroporphyrin. Significant increases in serum cholesterol and hepatic pentoxyresorufin dealkylase activity were observed in the highest-dose males and two highest-dose females. By contrast, liver UDP-glucuronosyl transferase activity was elevated in the two highest-dose males and the highest-dose females. Urinary ascorbic acid excretion was increased in the highest-dose males. While the amount of vitamin A was decreased dose-dependently, starting at 0.5 ppm in the liver of both sexes and in the lung of the females, the level in the kidney of the highest-dose group was increased. Administration of PCB 105 resulted in decreased dopamine in the caudate nucleus region of the brain in males and homovanillic acid in caudate nucleus and nucleus accumbens of females. Increased 5-hydroxytryptamine and 5-hydroxyindoleacetic acid were observed in the substantia nigra region of both sexes, with most of the increases being seen in highest-dose females. Anemia, characterized by decreased hemoglobin, hematocrit and red cell indices, occurred in the highest-dose group, as did eosinophilia. Treatment with PCB 105 caused dose-dependent histopathological changes in the liver and thyroid. Thymic changes were observed in the highest-dose males and two highest-dose females. Tissue residue data showed a dose-dependent accumulation of this congener in fat, liver and spleen, kidney and brain. Based on these data the no-observable-effect level of PCB 105 was judged to be 0.05 ppm or 3.9 microg kg(-1) body wt. day(-1) in males and 4.2 microg kg(-1) body wt. day(-1) in females.

Effect of phytochemicals on cytochrome P450-linked alkoxyresorufin O-dealkylase activity

Eight compounds of plant origin with potential chemopreventive properties were studied for their effects on hamster liver microsome-mediated alkoxyresorufin O-dealkylase activity. The phytochemicals investigated were (+)-catechin, chlorophyllin, crocetin, curcumin, diallyl sulphide, (-)-epigallocatechin gallate (EGCG), d-limonene and tannic acid. Each phytochemical at concentrations of 0.25 μM and 0.5 μM was incubated with 0.2 mg hamster liver microsomal protein and 0.5 μmole benzyloxyresorufin, methoxyresorufin and ethoxyresorufin. The resorufins serve as substrates for measurements of the activity of isoforms of cytochrome P450 (CYP). Benzyloxyresorufin O-dealkylase (BROD), methoxyresorufin demethylase (MROD) and ethoxyresorufin deethylase (EROD) are linked to CYP 2C11, CYP 1A2 and CYP 2C6, respectively. Chlorophyllin, curcumin, EGCG and tannic acid were all potent inhibitors of BROD, MROD and EROD activity. (+)-Catechin and diallyl sulphide inhibited BROD and EROD but not MROD activity. Crocetin and d-limonene inhibited BROD activity more than MROD and EROD. These results indicate that the enzymatic activity of cytochrome P450 isoforms 2C11, 1A2 and 2C6 can be altered by phytochemicals and that phytochemicals can affect the metabolism of substrates for these isoforms.

Characterization of the induction of rat microsomal cytochrome P450 by tacrine

The effect of multiple-dose tacrine (THA) administration at 2 and 20 mg/kg (single oral doses for 2 weeks) on cytochrome P450 (CYP) was examined in male and female Wistar rats. Changes in CYP were determined by measuring total spectral CYP, the rates of ethoxy- and pentoxyresorufin dealkylations, and the protein expression of several CYPs by western blot analysis of liver microsomes. Animals treated with β-naphthoflavone or phenobarbital were employed as positive controls. No physiological or metabolic changes were observed in male or female rats treated with 2 mg/kg THA for 2 weeks. Male and female animals treated with 20 mg/kg THA for 2 weeks demonstrated increased CYP1A activity (increased ethoxyresorufin deethylase activity) and increased expression of CYP1A1 with only minor increases in CYP1A2 expression. Compared with the effects of β-naphthoflavone induction of CYP1 A, the induction observed with THA at 20 mg/kg was considered minor.

SUBCHRONIC TOXICITY OF 2,2′,3,3′,4,4′-HEXACHLOROBIPHENYL IN RATS

The subchronic toxicity of 2,2′,3/3′,4,4′-hexachlorobiphenyl (PCB 128) was investigated in rats following dietary exposure at 0, 0.05, 0.5, 5, or 50 ppm for 13 wk. The growth rate was not affected by treatment and no apparent clinical signs of toxicity were observed. There was a significant increase in liver weight in the 50 ppm females. The liver ethoxy-resorufin deethylase (EROD) activity was increased by five- and fourfold in the highest dose males and females, respectively, while aminopyrine demethylase (ADPM) activity was significantly increased only in the highest dose females. Liver vitamin A was significantly reduced in the highest dose females. No other biochemical or hematological effects were observed. Treatment-related histopathological changes were seen in the thyroid and liver, and to a lesser extent in the bone marrow and thymus. Residue data showed a dose-dependent accumulation of PCB 128 in the following tissues: fat, liver, kidney, brain, spleen, and serum, with the highest concentration being found in fat followed by liver and kidney. Based on these data, the no-observable-adverse-effect level of PCB 128 was judged to be 0.5 ppm in diet or 42 yg/kg body weight.

SUBCHRONIC TOXICITY OF 2,2',3,3',4,4'-HEXACHLOROBIPHENYL IN RATS

The subchronic toxicity of 2,2{prime},3,3{prime},4,4{prime}-hexachlorobiphenyl (PCB 128) was investigated in rats following dietary exposure at 0, 0.05, 0.5, 5, or 50 ppm for 13 wk. The growth rate was not affected by treatment and no apparent clinical signs of toxicity were observed. There was a significant increase in liver weight in the 50 ppm females. The liver ethoxy-resorufin deethylase (EROD) activity was increased by five- and fourfold in the highest dose males and females, respectively, while aminopyrine demethylase (ADPM) activity was significantly increased only in the highest dose females. Liver vitamin A was significantly reduced in the highest dose females. No other biochemical or hematological effects were observed. Treatment-related histopathological changes were seen in the thyroid and liver, and to a lesser extent in the bone marrow and thymus. Residue data showed a dose-dependent accumulation of PCB 128 in the following tissues: fat, liver, kidney, brain, spleen, and serum, with the highest concentration being found in fat followed by liver and kidney. Based on these data, the no-observable-adverse-effect level of PCB 128 was judged to be 0.5 ppm in diet or 42 {mu}g/kg body weight. 29 refs., 1 fig., 5 tabs.

Effects of Benomyl on Cytochrome P450 Monooxygenase Systems in the Hep G2 Cell Line

Changes in the cytochrome P450 monooxygenase system were investigated in Hep G2 cells treated for 24 hours with various concentrations of benomyl. Decreases in both benzo[a]pyrene hydroxylase (AHH) and ethoxyresorufin deethylase (EROD), markers of the P4501A1 isoenzyme, were noted. Ethoxycoumarin deethylase (ECOD), a marker of the P4502B1 isoenzyme, showed a dose-dependent increase. Characterisation by SDS-polyacryl-amide gel electrophoresis of Hep G2 cell microsomal proteins revealed a decrease in the polypeptide bands at 55.5kD (P4501A1) and 48kD (P4501A1 and P4502B1) and an increase in the polypeptide band at 52kD (P4502B1). Benomyl induced a decrease in cytochrome P4501A1 and an increase in cytochrome P4502B1 in Hep G2 cells, as indicated by variations in AHH, EROD and ECOD activity, and by characterisation of microsomal proteins by SDS-polyacryl-amide gel electrophoresis.

Inducibility of ethoxyresorufin deethylase and UDP-glucuronosyltransferase activities in two human hepatocarcinoma cell lines KYN-2 and Mz-Hep-1.

Two human hepatoma cell lines, KYN-2 and Mz-Hep-1 were characterized in terms of glucuronidation capacity and inducibility of cytochrome P4501A1/1A2 and several UDP-glucuronosyltransferases (UGTs). Cytochrome P4501A1/1A2 activity was measured using 7-ethoxyresorufin and that of UGTs with 16 different substrates. The effects of dimethyl sulfoxide (DMSO), 3-naphthoflavone, alpha-naphthoflavone, and rifampicin on these drug-metabolizing enzyme activities were studied. DMSO treatment increased in a dose-dependent manner the ethoxyresorufin O-deethylase (EROD) activity in KYN-2 cells, while an opposite effect was observed in Mz-Hep-1 cells. In KYN-2 cells, EROD was more responsive toward beta-naphthoflavone treatment in combination with DMSO. This activity was enhanced in Mz-Hep-1 cells more than 83 times by beta-naphthoflavone. The enhancement of EROD activity by DMSO and beta-naphthoflavone treatments of KYN-2 cells was abolished by alpha-naphthoflavone treatment. In Mz-Hep-1, only the inducing effect of beta-naphthoflavone was abolished by alpha-naphthoflavone treatment. Rifampicin treatment of KYN-2 cells reversed both the DMSO and beta-naphthoflavone effects on the EROD activity. Glucuronidation of steroids, bile acids, fatty acids and drugs was effective in KYN-2 and Mz-Hep-1 cells. Both 1-naphthol glucuronidation and the level of UGT1*6 protein detected by immunoblot and supporting this activity were lowered by DMSO treatment and increased by beta-naphthoflavone treatment in KYN-2 cells. In Mz-Hep-1 cells, DMSO and beta-naphthoflavone had no effect on 1-naphthol glucuronidation activity. DMSO, beta-naphthoflavone and rifampicin also affected the glucuronidation of various substrates supported by different UGT isoforms. These results indicate that KYN-2 and Mz-Hep-1 cells can be used as new in vitro models for the studies of drug metabolism and the regulation of the corresponding enzymes.

Liver microsomal levels of cytochrome P450IA1 as biomarker for exposure and bioavailability of soil-bound polycyclic aromatic hydrocarbons

The bioavailability of soil-bound polycyclic aromatic hydrocarbons (PAHs) for mammalian species was studied with rats fed with a diet containing contaminated soil preparations. The extent of cytochrome P450IA1 (CYP1A1) induction in the liver correlated with the amount of 5- and 6-ring PAHs in the soil samples but not with the total PAH content. Other cytochromes P450 were much less affected by the soil-contaminants. The highest induction of CYP1A1 was obtained with a sample containing 274 mg 5- and 6-ring PAH/kg soil, resulting in a nearly 360-fold increase in the ethoxyresorufin deethylase (EROD) activity. In a semilogarithmic plot, a linear correlation was found between the 5- and 6-ring PAH concentration in the soil and the microsomal CYP1A1 content. As a model for the action of intestinal fluids, soil samples were extracted by bile acid solution. In these experiments, the selectivity in the solubilization of individual PAHs parallels that of toluene extraction, although the yield is lower than the latter and varies with the soil sample. The bioavailability of PAHs for microorganisms, but not for mammals, was shown to be considerably reduced in the presence of high total organic carbon (TOC) values of the soil samples. This may have implications for decontamination strategies, diminishing the effectiveness of biological decontamination in cases with high TOC values. The data suggest that CYP1A1 induction in rats is a parameter that may be useful in risk assessments of contaminated soils for mammalian species.

The effects of acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on glioma-specific cytotoxic T-cell activity in Fischer 344 rats

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is one of the most potent environmental immunomodulating agents identified so far. Historically, mice have been used to model mammalian immunobiology and most of the data gathered on the immunotoxicity of TCDD has been obtained from studies with mice. However, rats have been used more extensively in lexicological research to establish human risk assessment criteria. A need exists, therefore, to develop a database using the rat model in immunotoxicology so that complete animal toxicity studies can be conducted. We have treated female Fischer 344 rats with a single i.p. dose of 0.3,3.0, or 30.0 μ/kg TCDD or corn oil vehicle and examined cytotoxic T-cell (CTL) activities 24 days following treatment. Syngeneic in vivo tumor-specific CTLs were generated that model cell-mediated immune reactions against neoplastically transformed self antigens. RT2, a virally-induced Fischer 344 rat glioma, and D74, a ethylnitrosurea-induced Fischer 344 rat glioma were used as targets. This immunological parameter was compared to body, thymic, and liver weights as well as liver ethoxyresorufin deethylase (EROD) activity on day 24 post-TCDD treatment. The results indicate that Fischer 344 rats are very sensitive to TCDD as indicated by severe thymic atrophy and EROD induction at all three doses. In contrast, CTL activity was only marginally affected by these same doses of TCDD with only a modest suppression noted at the highest dose. These results indicate that the CTL response in rats may not be useful in characterizing the effects of this xenobiotic on immunocompetence in the rat.

Induction of rat liver drug-metabolizing enzymes by heterocycle-containing Mono-, Di-, Tri- and tetra-arylmethanes

The effect of a nitrogen heterocycle constituent on the ability of arylmethanes to induce phase I and phase II drug-metabolizing enzymes has been examined. Rats were treated with tetra-, tri-, di- or monoarylmethane compounds daily for 3 days at a dose of 75mg/kg. Induction of UDP- glucuronosyltransferase(morphine) activity was seen with twelve of the eighteen compounds investigated and for three compounds it occurred independent of any induction of cytochrome P450. Induction of glutathione S-transferase activity was seen with ten of the compounds and was generally paralleled by changes in overall cytochrome P450 concentration and in both pentoxyresorufin and erythromycin dealkylase activities. Major induction of ethoxyresorufin deethylase activity was only apparent with two diarylmethanes that contained a 1-substituted imidazole moiety. UDP-glucuronosyltransferase(1- naphthol) activity was coinduced by these two compounds. A tthird compound, diphenyl-4-pyridylmethane induced UDP-glucuronosyltransferase(1-naphthol) activity without increasing ethoxy-resorufin deethylase activity. Cytosolic sulfotransferase activity was not induced by the administration of any compound in this study. Among arylmethane derivatives, the presence of two aryl groups appeared to be a minimum requirement for induction of drug-metabolizing enzymes. If one of the aryl groups was not a heterocycle, or if the nitrogen atom of the heterocycle was sterically hindred, major induction of cytochrome P450 did not occur. With triarylmethanes, induction was independent of whether the heterocycle was imidazole, pyridine or pyrimidine.

Cytochrome P450 in trypanosomatids

Post-mitochondrial supernatant extracts prepared from bloodstream forms of Trypanosoma brucei brucei, T. cruzi epimastigotes, Leishmania donovani promastigotes and Crithidia fasciculata have been found to catalyse cytochrome P450-dependent reactions. Appreciable ethoxycoumarin deethylase and ethoxyresorufin deethylase activities were found in all of the above trypanosomatids, with T. cruzi epimastigotes having the highest activity (57.1 and 10.7 pmol/min/mg protein, respectively). In all four species these reactions were inhibited by the cytochrome P450 inhibitors carbon monoxide, proadifen and metyrapone. In contrast to rat liver microsomes, the trypanosomatid extracts showed no detectable pentoxyresorufin depentylase or pentamidine hydroxylase activity. Both C. fasciculata and T. b. brucei post-mitochondrial supernatants showed carbon monoxide difference spectra consistent with the presence of cytochrome P450 (9.6 and 6.3 pmol/mg protein, respectively). An additional hemoprotein which gave a carbon monoxide difference peak at 420 nm was also detected in C. fasciculata and T. b. brucei microsomes and C. fasciculata mitochondria. Subcellular fractionation of both early and late log C. fasciculata showed that the ethoxycoumarin deethylase activity was enriched in the microsomal fraction.

N-benzylimidazole-mediated changes in hepatic drug-metabolizing enzyme activities in Ah-responsive and Ah-non-responsive mice.

1. The inductive effect of N-benzylimidazole (NBI) on hepatic microsomal and cytosolic drug-metabolizing enzyme activities in aryl hydrocarbon (Ah)-responsive C57BL/6N (B6) and Ah-non-responsive DBA/2N (D2) mouse strains was determined and compared with that caused by beta-naphthoflavone (BNF). 2. Relative Ah-responsiveness of the two strains was confirmed by measurement of BNF-induced ethoxyresorufin deethylase (EROD) activity and ELISA immunoquantification. BNF markedly induced EROD activity only in the Ah-responsive B6 mouse strain (65-fold increase). 3. NBI (150 mg/kg per day for 3 days) increased cytochrome P450 concentration similarly in both strains (40 and 60% in B6 and D2 strains, respectively). Compared with BNF treatment of the B6 strain, increases in EROD activity following NBI treatment were only minor. In addition, EROD activity increases were greater in the Ah-nonresponsive D2 strain (300%) than in the Ah-responsive B6 strain (100%) suggesting the possibility of an induction mechanism different from that of recognized Ah receptor agonists. 4. Induction of UDP-glucuronosyltransferase activity (p-nitrophenol acceptor) by BNF was greater in the Ah-responsive B6 strain than in the Ah-non-responsive D2 strain. NBI failed to induce this activity in either strain. 5. Induction of glutathione S-transferase activity towards 1-chloro-2,4-dinitrobenzene following NBI treatment occurred to the same extent (25% increase) as that seen following BNF treatment, in the Ah-responsive B6 strain. Neither xenobiotic affected this activity in the Ah-non-responsive D2 strain. 6. Although NBI is a major inducer, possessing Ah-like inducing properties in rat, it caused only minor changes in murine drug metabolizing enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of ethanol on microsomalp‐nitrophenol hydroxylation and ethoxyresorufin deethylation in rat liver and lung

The ability of ethanol to induce the deethylation of ethoxyresorufin and hydroxylation of p-nitrophenol in rat lung and liver was investigated. The administration of 10% ethanol in the drinking water for 7 d increased hepatic p-nitrophenol hydroxylation twofold whereas ethanol ip (3 ml/kg/d for 7 d) decreased the activity. Both ethanol treatments decreased the activity in the lung. Ethoxyresorufin deethylation was induced in the liver by both ethanol treatments, and in the lung the bolus dosing ip increased the activity four-fold. The results indicate that the lung microsomes have p-nitrophenol hydroxylase and ethoxyresorufin deethylase activities, but only the latter was increased by ethanol treatment.