Hepatic Microsomal Enzyme Activity Research Articles

Mode of action analysis for fluxapyroxad-induced rat liver tumour formation: evidence for activation of the constitutive androstane receptor and assessment of human relevance

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000 ppm fluxapyroxad for 3 and 7 days and in female rats given 50–3000 ppm fluxapyroxad for 7 days and 250–3000 ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250–3000 ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1–100 μM fluxapyroxad or 500 μM sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid β-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25 ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1–100 μM fluxapyroxad or 500 μM NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1–16 μM fluxapyroxad or 500 μM NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.

LBODP105 A Case of Significantly Increased Levothyroxine Requirement With Rifampin Therapy

Abstract Introduction Rifampin has a wide range of drug interactions through increased hepatic microsomal enzyme activity. We are reporting a case of a patient with post-surgical hypothyroidism on stable thyroid hormone replacement therapy who required significantly higher doses of levothyroxine after starting rifampin. Case Report Patient is a 78-year-old woman with history of Papillary Thyroid Cancer (PTC) in remission status post thyroidectomy in 1994 and radioactive iodine therapy in 1997. Patient had been on a stable dose of Levothyroxine of 75-88mcg daily until a visit in January 2013 when her TSH was found to be 39 despite compliance with levothyroxine. Further history revealed that she was diagnosed with Mycobacterium Avium Intracellulare (MAI) infection of the lungs for which she was given rifampin therapy. Her Levothyroxine dose was progressively increased to 150mcg daily. She received rifampin therapy for 18 months, from August 2012 until April 2013. Her Levothyroxine dose was later changed to average 139 mcg /day based on TSH levels and continued to be the same through the rifampin course. In April 2014, and following discontinuation of rifampin, Levothyroxine was decreased to 100mcg daily and subsequently 75mcg daily. During a relapse of MAI in March 2016, her levothyroxine dosage was similarly again increased to 125mcg daily for TSH of 24.58. In June 2017, rifampin was discontinued after completion of second course of MAI treatment, and Levothyroxine dose decreased to 88 mcg daily, and subsequently 75 mcg in September 2017, and average daily 64mcg most recently given age and long-term remission with target mid normal TSH. Discussion Rifampin is used to treat active tuberculosis and MAI infection among other uses. It is a hepatic enzyme inducer which significantly interacts with other drugs. It has been reported previously to cause hypothyroidism in patients with underlying thyroid disorder and increased serum TSH while on levothyroxine, but its impact range on thyroid function has not been clearly described. Our patient was on stable levothyroxine dose following thyroidectomy, and required almost a 100% increase of levothyroxine dose when started on rifampin therapy, returning to baseline levothyroxine requirements off rifampin. This case highlights the severe hypothyroidism and significant impact on levothyroxine dose requirements following rifampin therapy, and the importance of close thyroid function monitoring in patients with hypothyroidism and on rifampin, especially in context of post-surgical hypothyroidism. Presentation: No date and time listed

Extraction of Ethyl-p-methoxycinnamate from Kaempferia galanga L.

In this study, ethyl-p-methoxycinnamate (EPMC) was extracted from Kaempferia galanga L. by different methods. The study results show that vacuum drying process led to a significant decrease in EPMC content in the herb (about 7%); EPMC obtained by water distillation not only had a high purity and yield but could also be applied for both fresh and dried herb; whereas, Soxhlet extraction with n-hexane solvent brought the highest extraction yield (1.99% on dried herb weight) despite lower purity when compared to that by water distillation method.
 Keywords
 Ethyl-p-methoxycinnamate, EPMC, Kaempferia galanga L., extraction.
 References
 [1] Botany Department, Botany, Hanoi University of Pharmacy, 2005 (In Vietnamese).[2] A. Kumar, Phytochemistry, pharmacological activities and uses of Indian traditional medicinal plant Kaempferia galanga L. – An overview, Journal of Ethnopharmacology (2020) 112667. https://doi.org/10.1016/j.jep.2020.112667.[3] H.J. Ko, H.J. Kim, S.Y. Kim, H.Y. Yun, K.J. Baek, N.S. Kwon, W.K. Whang, H.R. Choi, K.C. Park, D.S. Kim, Hypopigmentary Effects of Ethyl P‐Methoxycinnamate Isolated from Kaempferia galanga, Phytotherapy research 28(2) (2014) 274-279. https://doi.org/10.1002/ptr.4995.[4] M. Bonesi, J. Xiao, R. Tundis, F. Aiello, V. Sicari, M. R. Loizzo, Advances in the Tyrosinase Inhibitors from Plant Source, Current Medicinal Chemistry 26(18) (2019) 3279-3299. https://doi.org/10.2174/0929867325666180522091311.[5] M.I. Umar, M.Z. Asmawi, A. Sadikun, A.M. Majid, F.S. Al-Suede, L.E. Hassan, R. Altaf, M.B. Ahamed, Ethyl-p-methoxycinnamate isolated from Kaempferia galanga inhibits inflammation by suppressing interleukin-1, tumor necrosis factor-α, and angiogenesis by blocking endothelial functions, Clinics 69(2) (2014) 134-144. https://doi.org/10.6061/clinics/2014(02)10.[6] D. Lakshmanan, J. Werngren, L. Jose, K.P. Suja, M.S. Nair, R.L. Varma, S. Mundayoor, S. Hoffner, R.A. Kumar, Ethyl p-methoxycinnamate isolated from a traditional anti-tuberculosis medicinal herb inhibits drug resistant strains of Mycobacterium tuberculosis in vitro, Fitoterapia 82(5) (2011) 756-761. https://doi.org/10.1016/j.fitote.2011.03.006.[7] W. Sirisangtragul, B. Sripanidkulchai, Effects of Kaempferia galanga L. and ethyl-p-methoxycinnamate (EPMC) on hepatic microsomal cytochrome P450s enzyme activities in mice, J. Sci. Technol. 33(4) (2011) 411-417.[8] J. Wu, F. Ge, D. Wang, X. Xu, Combination of supercritical fluid extraction with high-speed countercurrent chromatography for extraction and isolation of ethyl p -methoxycinnamate and ethyl cinnamate from Kaempferia galanga L., Separation Science and Technology 51(10) (2016) 1757-1764. https://doi.org/10.1080/01496395.2016.1176046.[9] N. Srivastava, Ranjana, S. Singh, A.C. Gupta, K. Shanker, D.U. Bawankule, S. Luqman, Aromatic ginger (Kaempferia galanga L.) extracts with ameliorative and protective potential as a functional food, beyond its flavor and nutritional benefits, Toxicol Rep 6 (2019) 521-528. https://doi.org/10.1016/j.toxrep.2019.05.014.[10] I. Komalaa, Supandia, Nurhasnib, O.S. Bethaa, Yardia, S. Mufidaha, M. Rezaa, M.S. Alia, N.S. Auliaa, Sutara, Microwave Assisted Synthesis of p-Methoxycinnamamides and p-Methoxy-β-nitrostyrenes from Ethyl p-methoxycinnamate and Screening their Anti-inflammatory Activity, Natural Product Communications 12(8) (2017) 1265-1268.[11] Vietnamese Pharmacopoeia, the 5th edition, Appendix 2, (2017) (In Vietnamese)[12] AOAC International, AOAC official methods of analysis, Appendix K: Guidelines for Dietary Supplements and Botanicals, 2013.[13] P. Lunger, M. Weber, N. X. Dung, N. T. B. Tuyet, Ethyl p-Methoxycinnamate from Kaempferia galanga L. in Vietnam, Acta Cryst C52 (1996) 1255-1257. https://doi.org/10.1107/S0108270195016027.[14] S.K. Gupta, A.B. Banerjee, Achari B., Isolation of Ethyl p-methoxycinnamate, the major antifungal principle of Curcumba zedoaria, Curcumba zedoaria. Lloydia 39(4) (1976) 218-222.[15] M.I. Umar, M.Z. Asmawi, A. Sadikun, I.J. Atangwho, M.F. Yam, R. Altaf, A. Ahmed, Bioactivity-guided isolation of ethyl-p-methoxycinnamate, an anti-inflammatory constituent, from Kaempferia galanga L. extracts, Molecules 17(7) (2012) 8720-34. https://doi.org/10.3390/molecules17078720.

The role of acid-base imbalance in statin-induced myotoxicity

Disturbances in acid-base balance, such as acidosis and alkalosis, have potential to alter the pharmacologic and toxicologic outcomes of statin therapy. Statins are commonly prescribed for elderly patients who have multiple comorbidities such as diabetes mellitus, cardiovascular, and renal diseases. These patients are at risk of developing acid-base imbalance. In the present study, the effect of disturbances in acid-base balance on the interconversion of simvastatin and pravastatin between lactone and hydroxy acid forms have been investigated in physiological buffers, human plasma, and cell culture medium over pH ranging from 6.8–7.8. The effects of such interconversion on cellular uptake and myotoxicity of statins were assessed in vitro using C2C12 skeletal muscle cells under conditions relevant to acidosis, alkalosis, and physiological pH. Results indicate that the conversion of the lactone forms of simvastatin and pravastatin to the corresponding hydroxy acid is strongly pH dependent. At physiological and alkaline pH, substantial proportions of simvastatin lactone (SVL; ∼87% and 99%, respectively) and pravastatin lactone (PVL; ∼98% and 99%, respectively) were converted to the active hydroxy acid forms after 24 hours of incubation at 37°C. At acidic pH, conversion occurs to a lower extent, resulting in greater proportion of statin remaining in the more lipophilic lactone form. However, pH alteration did not influence the conversion of the hydroxy acid forms of simvastatin and pravastatin to the corresponding lactones. Furthermore, acidosis has been shown to hinder the metabolism of the lactone form of statins by inhibiting hepatic microsomal enzyme activities. Lipophilic SVL was found to be more cytotoxic to undifferentiated and differentiated skeletal muscle cells compared with more hydrophilic simvastatin hydroxy acid, PVL, and pravastatin hydroxy acid. Enhanced cytotoxicity of statins was observed under acidic conditions and is attributed to increased cellular uptake of the more lipophilic lactone or unionized hydroxy acid form. Consequently, our results suggest that comorbidities associated with acid-base imbalance can play a substantial role in the development and potentiation of statin-induced myotoxicity.

Epigenetic effects of dietary butyrate on hepatic histone acetylation and enzymes of biotransformation in chicken

The aim of the study was to investigate the in vivo epigenetic influences of dietary butyrate supplementation on the acetylation state of core histones and the activity of drug-metabolising microsomal cytochrome P450 (CYP) enzymes in the liver of broiler chickens in the starter period. One-day-old Ross 308 broilers were fed a starter diet without or with sodium butyrate (1.5 g/kg feed) for 21 days. After slaughtering, nucleus and microsome fractions were isolated from the exsanguinated liver by multi-step differential centrifugation. Histone acetylation level was detected from hepatocyte nuclei by Western blotting, while microsomal CYP activity was examined by specific enzyme assays. Hyperacetylation of hepatic histone H2A at lysine 5 was observed after butyrate supplementation, providing modifications in the epigenetic regulation of cell function. No significant changes could be found in the acetylation state of the other core histones at the acetylation sites examined. Furthermore, butyrate did not cause any changes in the drugmetabolising activity of hepatic microsomal CYP2H and CYP3A37 enzymes, which are mainly involved in the biotransformation of most xenobiotics in chicken. These data indicate that supplementation of the diet with butyrate probably does not have any pharmacokinetic interactions with simultaneously applied xenobiotics.

Moduratory effect of Thai traditional medicine (Yahom Tultavai) on hepatic cytochrome P450 enzymes and pentobarbital-induced sleeping in mice

Yahom Tultavai is a Thai traditional medicine that has been widely used for the treatment of nausea, vomiting, dizziness and weakness in aged-people, especially. Its formula contains several medicinal plants, and one of them is Kaempferia galanga L., which has ethyl-p-methoxycinnamate (EPMC) as its major compound. Recently, several herbs and traditional medicines have been reported to demonstrate herbal-drug interaction with conventional medicines. This study aims to investigate the effect of Yahom Tultavai extracts on hepatic cytochrome P450 enzymes and pentobarbital-induced sleeping in mice. Three extracts of Yahom Tultavai, using dichloromethane, methanol and distilled water as solvents were orally administered for 28 days prior to determine CYP1A1, CYP1A2, CYP2B, CYP2E1 and CYP3A4 activities. All three extracts significantly inhibited CYP1A1, CYP1A2 and CYP 2E1 activities, but only dichloromethane extract enhanced CYP2B activity. In addition, all three extracts had no effect on CYP3A4 activity. As an indicator for metabolic drug interaction, pentobarbital-induced sleeping time was decreased in connection with the induction of CYP2B activity between 7 and 28 days of dichloromethane extract and EPMC-treated animals when compared to control. In conclusion, Yahom Tultavai extracts affected hepatic microsomal CYP enzyme activities and reduced pentobarbital-induced sleeping time in mice. The results suggest that Yahom Tultavai may potentially cause herbal and conventional drug interaction, which can affect the clinical implication of drug action. Therefore, the co-administration of Yahom Tultavai with certain drugs should be carefully considered.

Identification of Diacylglycerol Acyltransferase Inhibitors from Rosa centifolia Petals

Diacylglycerol acyltransferase (DGAT) catalyzes the final step of triacylglycerol (TAG) synthesis, and is considered as a potential target to control hypertriglyceridemia or other metabolic disorders. In this study, we found that the extract of rose petals suppressed TAG synthesis in cultured cells, and that the extract showed DGAT inhibitory action in a dose-dependent manner. Fractionation of the rose extract revealed that the DGAT inhibitory substances in the extract were ellagitannins; among them rugosin B, and D, and eusupinin A inhibited DGAT activity by 96, 82, and 84% respectively, at 10μM. These substances did not inhibit the activities of other hepatic microsomal enzymes, glucose-6-phosphatase and HMG-CoA reductase, or pancreatic lipase, suggesting that ellagitannins inhibit DGAT preferentially. In an oral fat load test using mice, postprandial plasma TAG increase was suppressed by rose extract; TAG levels 2h after the fat load were significantly lower in mice administered a fat emulsion containing rose extract than in control mice (446.3±33.1 vs 345.3±25.0mg/dL, control vs rose extract group; P<0.05). These results suggest that rose ellagitannins or rose extract could be beneficial in controlling lipid metabolism and used to improve metabolic disorders.

Development of an optimized procedure for the preparation of rat intestinal microsomes: comparison of hepatic and intestinal microsomal cytochrome P450 enzyme activities in two rat strains

The objective of this study was to characterize cytochrome P450 (CYP) activities in both intestinal and hepatic microsomes from Wistar and Sprague–Dawley rats.Specific probes for measuring CYP activities were selected using rat recombinant CYP.The intestinal microsome preparation was optimized getting a more relevant and reproducible abundance of CYPs to measure CYP activities.Testosterone, propranolol, diclofenac, and midazolam were determined as specific substrates of rat CYP2C11, CYP2D2, CYP2C6, and CYP3A, respectively. Ethoxyresorufin and pentoxyresorufin were not specific substrates of CYP1A2 and CYP2B1, respectively. Hepatic and intestinal microsomes expressed active CYP1A1, CYP1A2, CYP2B1, and CYP3A2. Only liver expressed active CYP2C6, CYP2C11, and CYP2D2. Wistar liver expressed more active CYP1A and CYP3A2, but less active CYP2B1 than Wistar intestine. Sprague–Dawley liver expressed more active CYP2B1 and CYP3A2, but less active CYP1A than Sprague–Dawley intestine.In conclusion, CYP activities were qualitatively equivalent but not quantitatively in both strains.

Rifampin-Induced Hypothyroidism without Underlying Thyroid Disease

Rifampin (RFP) increases hepatic microsomal enzyme activity, and there are case reports of RFP-induced hypothyroidism, all associated with Hashimoto's thyroiditis. Here, we report a case of RFP-induced hypothyroidism without underlying thyroid disease.

Long-term exposure of European flounder ( Platichthys flesus) to the flame-retardants tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD)

Tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) are widely used flame retardants that have increasingly been found as contaminants in the aquatic environment. In the present study, European flounder ( Platichthys flesus) were chronically exposed to TBBPA; (105 days) and HBCD (78 days), in a wide range including environmentally relevant concentrations. TBBPA was administered via the water, whereas HBCD was administered in food and sediment, or in sediment alone. Chemical analysis of muscle showed an average increase in internal concentrations of approximately two orders of magnitude for both compounds tested. Animals exposed to HBCD via sediment alone (8000 μg/g total organic carbon, TOC) showed a proportional increase of α-HBCD in muscle compared to animals exposed via food and sediment. In both studies, exposure to the test compounds did not affect general health and toxicity parameters (behavior, survival, growth rate, relative liver and gonad weight). Hepatic microsomal enzyme activities (TBBPA: EROD; HBCD: EROD, PROD, and BROD) were not induced by any of the tested chemicals. Aromatase activity in male gonads showed a mild increase with rising TBBPA levels. There were no morphological and immunohistochemical indications for increased production of the yolk precursor protein vitellogenin (VTG) in animals exposed to TBBPA and HBCD; immunochemical analysis of plasma VTG levels showed no dose response in animals exposed to TBBPA. In animals exposed to TBBPA, levels of the thyroid hormone thyroxin (T 4) increased with internal concentrations of the test compound, possibly indicating competition of TBBPA for plasma protein binding. Triiodothyronin (T 3) levels were not affected and histology showed no signs of altered thyroid gland activity. Other organs investigated (liver, gills, kidney, skin, and gonads) revealed no histological changes related to TBBPA or HBCD exposure. Overall, the present results indicate limited endocrine effects of these widely used flame retardants in a test species representative of European estuaries at environmentally relevant exposure levels and at internal levels up to 4300 ng TBBPA/g wet weight, and 446 μg HBCD/g lipid weight in flounder muscle.

DOSE- AND TIME-DEPENDENT EFFECTS OF 2,3,7,8-TETRABROMODIBENZO-P-DIOXIN ON RAT LIVER

Dose- and time-dependent effects of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) on the liver were examined by single administration of TBDD by gavage to male and female rats. Fifteen Wistar rats of each sex per group received 0, 10, 30, 100 or 300 microg TBDD/kg body weight. Rats surviving to scheduled necropsy on Day 2, 7 or 36 after the TBDD administration were examined for hepatic histopathology, activities of hepatic microsomal enzymes and serum levels of lipids, total cholesterol and transaminases and hepatic concentrations of TBDD. Tigroid basophilic cytoplasm and hepatocellular hypertrophy were observed at 10 microg/kg on Day 2 or 7 through 36, whereas degenerative and aggressive lesions such as necrosis, fibrosis, multinucleated hepatocytes and disarrangement of hepatocytes occurred later at higher dose levels. Persistently increased activities of hepatic aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD), increased serum levels of total cholesterol and phospholipid and increased relative liver weight were observed in all groups dosed 10 mug/kg and above, suggesting that hepatic microsomal monooxygenases and basophilic cytoplasm of hepatocytes were early and sensitive indicators among those TBDD-induced effects. A dose-dependent increase in liver concentrations of TBDD on Day 2 was followed by logarithmic decreases in TBDD concentrations against the days elapsed after the TBDD administration. An elimination half-life (t(1/2)) of TBDD from the liver was estimated to range from 12 to 16 days. It was suggested that females were more susceptible to TBDD than males, and that acute hepatotoxicity of TBDD was as potent as that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

In Vitro Inhibition of Rat CYP1A1 and CYP1A2 by Piceatannol, a Hydroxylated Metabolite of trans-Resveratrol

Piceatannol and its parent compound, trans-resveratrol, decreased the in vitro catalytic activity of rat CYP1A1 and CYP1A2 by mixed inhibition. trans-Resveratrol was not a mechanism-based inactivator of rat CYP1A in vitro and the administration of this compound (50 mg/kg) to rats did not affect hepatic microsomal CYP1A-mediated enzyme activity.

Effects of two infectious bursal disease vaccine virus strains on hepatic microsomal enzyme activities in chickens

The influence of two infectious bursal disease vaccines on the activities of hepatic microsomal enzymes aniline hydroxylase, ethylmorphine N-demethylase, NADPH-cytochrome c reductase, aryl sulphotransferase and p-nitrophenol UDP-glucuronyltransferase was investigated in chickens. The vaccines contained attenuated Winterfield 2512 and VMG-91 strains, respectively. The activities of enzymes were determined on postvaccination days 0, 2, 5 and 7. At the same time, post-mitochondrial supernatant, cytosolic and microsomal pellet protein concentrations were determined. As expected, the antibody titres against infectious bursal disease virus in the serum were increased in both tested groups in relation to each administered vaccine. Using RT-PCR, the presence of the VP2 gene fragment of virus in the liver of chicken was demonstrated 4 and 6 h after vaccination. The results of this study suggest that the two commercial vaccines modulate the activities of five enzymes tested, and that the two attenuated vaccines applied triggered induction and/or inhibition of phases I and II of biotransformation enzyme activities.

Species difference in the induction of hepatic CYP1A subfamily enzymes, especially CYP1A2, by 2-methoxy-4-nitroaniline among rats, mice, and guinea pigs

Species difference in the induction of hepatic cytochrome P450 CYP1A subfamily enzymes by 2-methoxy-4-nitroaniline (2-MeO-4-NA) was investigated among male F344 rats, C57BL/6 Cr mice, and Hartley guinea pigs. All species of animals were treated with a single ip injection of 2-MeO-4-NA (0.44 mmol/kg body weight), and changes in levels of the mRNA and protein of hepatic cytochrome P4501A (CYP1A) subfamily enzymes were examined by the methods of RT-PCR and Western blot, respectively. In addition, hepatic microsomal enzyme activities were measured using methoxyresorufin and ethoxyresorufin as substrates of CYP1A2 and CYP1A1, respectively. The overall results of the RT-PCR, Western blot, and measurement of the enzyme activity indicated that 2-MeO-4-NA-mediated induction of hepatic CYP1A subfamily enzymes, especially CYP1A2, occurred only in rats but not any other species of animals examined and that the species difference in the CYP1A induction was not necessarily correlated with that in pharmacokinetics of 2-MeO-4-NA. Furthermore, a luciferase reporter gene assay for screening of the ligands of arylhydrocarbon receptor (AhR) using a rat hepatic cell line suggested that 2-MeO-4-NA is not an AhR ligand. The present findings demonstrate for the first time the species difference in the 2-MeO-4-NA-mediated induction of hepatic CYP1A subfamily enzymes between rats and other rodents, mice and guinea pigs, and further propose an AhR-independent pathway for 2-MeO-4-NA-mediated induction in rats.

Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus)

Among other factors, cytochrome P450 (CYP) enzyme activity determines polychlorinated biphenyl (PCB) bioaccumulation, biotransformation, and toxicity in exposed species. We measured the oxidative metabolism in vitro of 12 PCB congeners, representing structural groups based on the number and position of the chlorine atoms, by the hepatic microsomes of one Baltic grey seal (Halichoerus grypus). Microsomal metabolism was observed for several PCBs with vicinal H atoms exclusively in the ortho and meta positions and without any ortho-Cl substituents (CB-15 [4,4'-Cl2] and CB-77 [3,3',4,4'-Cl4]), vicinal meta and para-H atoms (CB-52 [2,2',5,5'-Cl4], and -101 [2,2',4,5,5'-Cl5]) or with both characteristics in combination with either only one ortho-Cl (CB-26 [2,3',5-Cl3], CB-31 [2,4',5-Cl3]) or two ortho-Cl substituents (CB-44 [2,2',3,5'-Cl4]). To allocate PCB biotransformation to specific CYPs, the inhibitive effect of compounds with known CYP-specific inhibition properties was assessed on in vitro PCB metabolism and on regio- and stereospecific testosterone hydroxylase activities. Metabolic inhibition was considered relevant at concentrations < or = 1.0 microM because these inhibitors became decreasingly selective at higher concentrations. At < 1.0 microM, ellipticine (CYPIAI/2 inhibitor) selectively inhibited CB-15, -26, -31, and -77 metabolism, with no significant inhibition of CB-44, -52, and -101 metabolism. Inhibition of CB-52 and -101 metabolism by chloramphenicol (CYP2B inhibitor) started at 1.0 microM and maximized at about 100% at 10 microM. Ketoconazole (CYP3A inhibitor) appeared to selectively inhibit CB-26, -31, and -44 metabolism relative to CB-15, -77, and -52 at concentrations < or = 1.0 microM. Major testosterone metabolites formed in vitro were 2beta-(CYP3A), 6beta- (CYP3A, CYPIA), and 16beta- (CYP2B) hydroxytestosterone and androstenedione (CYP2B, CYP2C11). The CYP forms indicated are associated with the specific metabolism of testosterone in laboratory animals. Inhibition of 2beta- and 6beta-hydroxytestosterone formation at ellipticine and ketoconazole concentrations < or = 1.0 microM suggested that both inhibitors were good substrates of CYP3A-like enzymes in grey seal. Chloramphenicol (model for CYP2B) is apparently not a good inhibitor of CYPI A and CYP3A activities in grey seal because the chemical did not inhibit any metabolic route of testosterone at concentrations from 0.1 to 10 microM. Our findings demonstrated that at least CYP1A- and CYP3A-like enzymes in the liver of grey seals are capable of metabolizing PCBs with ortho-meta and/or meta-para vicinal hydrogens. A CYP2B form might also be involved, but this could not be proven by the results of our experiments. Defining the profiles of CYP enzymes that are responsible for PCB biotransformation is necessary to fully understand the bioaccumulation, toxicokinetics, and risk of PCB exposure in seals and other free-ranging marine mammals.

Potentiation of carbon tetrachloride hepatotoxicity by pentosan polysulfate in rats.

Few data are available in the literature regarding the effect of pentosan polysulfate (PPS) on normal and fibrotic rat livers. In addition, the combination of PPS and carbon tetrachloride (CCl4) has not been studied so far. The objective of this study was to assess the effect of PPS on rat livers treated or not with CCl4 for the induction of liver fibrosis. The study consisted of four stages: 1) hepatic fibrosis induction with CCl4 (N = 36 rats); 2) evaluation of the effect of PPS on CCl4-induced hepatic fibrosis (N = 36 rats); 3) evaluation of the effect of higher doses of PPS in combination with CCl4 (N = 50 rats); 4) evaluation of the presence of an enzymatic inductor effect by PPS (N = 18 rats) using the sodium pentobarbital test which indirectly evaluates hepatic microsomal enzyme activity in vivo. Adult (60 to 70 days) male Wistar rats weighing 180 to 220 g were used. All animals receiving 0.5 ml 8% CCl4 (N = 36) developed hepatic fibrosis, and after 8 weeks they also developed cirrhosis. No delay or prevention of hepatic fibrosis was observed with the administration of 5 mg/kg PPS (N = 8) and 1 mg/kg PPS (N = 8) 1 h after the administration of CCl4, but the increased hepatotoxicity resulting from the combination of the two substances caused massive hepatic necrosis in most rats (N = 45). PPS (40 mg/kg) alone caused hepatic congestion only after 8 weeks, but massive hepatic necrosis was again observed in association with 0.5 ml CCl4 after 1 to 4 weeks of treatment. Unexpectedly, sleeping time increased with time of PPS administration (1, 2, or 3 weeks). This suggests that PPS does not function as an activator of the hepatic microsomal enzymatic system. Further studies are necessary in order to clarify the unexpected increase in hepatotoxicity caused by the combination of CCl4 and high doses of PPS, which results in massive hepatic necrosis.

Persistent organochlorine residues and their bioaccumulation profiles in resident and migratory birds from North Vietnam

Concentrations of persistent organochlorines (OCs), such as polychlorinated biphenyls (PCBs), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and its metabolites (DDTs), hexachlorocyclohexane isomers (HCHs), hexachlorobenzene (HCB), and chlordane compounds (CHLs), were determined in whole-body homogenates of resident and migratory birds collected from the Red River estuary, North Vietnam, during March and October 1997. Contamination pattern was in the order of DDTs > PCBs > HCHs > CHLs > HCB in both resident and migratory birds. Residue concentrations, according to the feeding habit, showed little variability, which may reflect relatively similar trophic levels of the bird species analyzed. Resident birds accumulated greater concentrations of DDTs as compared to migrants. In contrast, HCH residues were greater in migratory species. Higher proportions of p,p'-DDT to total DDT concentrations were found in many species of residents and migrants, indicating recent exposure to technical DDT in northern Vietnam. Congener-specific PCB analysis showed the predominance of penta- and hexachlorobiphenyls in all the species analyzed. Estimation of hepatic microsomal enzyme activities suggested higher metabolic capacity for PCB congeners in shore birds from Vietnam as compared to higher-trophic predator birds and marine mammals. Comparison of OC residues in avian species in Asia-Pacific revealed that DDT residues in resident birds in North Vietnam are among the highest values reported for the countries surveyed, suggesting recent usage of DDT in Vietnam. Available data for birds, fish, and bivalves from the recent Asia-Pacific Mussel Watch Program suggested that Vietnam might be a potential source of DDT contamination in Asian developing countries. To our knowledge, this is the first study of the OC accumulation in avian species from Vietnam.

P450 in wild animals as a biomarker of environmental impact

The impact of environmental pollution on selected animals was tested by monitoring the hepatic content of cytochromes P450 and their enzyme activities or by calculating TEQ values from the concentration of pollutants in the body. Fish-eating Stellars Sea Eagles, Haliaeetus pelagicus, found dead in the northern part of Hokkaido island accumulated high levels of PCBs and DDT and metabolites. The TEQ values calculated from the PCB concentration in the eagles were high enough to cause a significant toxic effect in other birds living in the same environment. Some of these birds were also contaminated with high concentrations of lead. Spotted seals, Phoca largha, captured along the coast-line of Hokkaido accumulated PCBs in their fat at about 100 million times the concentrations in the surface sea water. The levels of expressions of hepatic microsomal CYP 1A1and related enzyme activities in these seals showed good correlation to the levels of PCBs accumulated in the fat. The fresh water crabs, Eriocheir japonicus, were captured from three different rivers with various degrees of pollution. The P450 content and the related enzyme activities showed good correlation to TEQ values obtained from the concentrations of PCBs and PCDDs in the crabs from the rivers. The wild rodents, Clethrionomys rufocanus, were captured from urban, agricultural, and forest areas in Hokkaido. Those from the forest area had the lowest CYP content and related enzyme activities, comparable to those in laboratory-raised animals. Those from the urban areas, presumably contaminated with PAHs from fuel combustion, showed increased CYP 1A1 content and related enzyme activities. Those from the agricultural areas showed increased levels of CYP 1A1, 2B, 2E1. Rats treated with some of the agrochemicals used in the area resulted in a similar pattern of induction. It is concluded that P450 can be a useful biomarker for assessing the environmental impact of chemical pollutants on wild animals.

DIELDRIN INDUCES CYTOSOLIC [3H]7,12-DIMETHYLBENZ[a]ANTHRACENE BINDING BUT NOT MULTIDRUG RESISTANCE PROTEINS IN RAINBOW TROUT LIVER

Previously it was demonstrated that biliary excretion of a single dose of [14C]dieldrin or [3H]7,12-dimethylbenz[a]anthracene (DMBA) was stimulated up to 700% and 300%, respectively, in rainbow trout fed 0.3?0.4 mg dieldrin/kg/d for 9?12 wk. This was not explained by increased activities of hepatic microsomal xenobiotic-metabolizing enzymes or increased amounts of any of six cytochrome P-450 isozymes quantitated by Western blots. It was hypothesized that stimulated excretion was explained by induction of (1) cytosolic binding proteins that facilitated intracellular trafficking of DMBA to sites of metabolism, or (2) ATP-dependent proteins that transport xenobiotic metabolites from liver to bile. Binding of 15 and 60 nmol [3H]DMBA/mg protein increased about 200% in hepatic cytosol from dieldrin-fed fish. A 50-fold molar excess of unlabeled DMBA reduced binding of 15 nmol [3H]DMBA/mg protein (nonspecific binding) by the same amount in cytosol from control and dieldrin-fed fish, indicating that dieldrin induced specific binding. Liver sections from control and dieldrin-fed fish were treated with multidrug resistance (MDR) protein monoclonal antibodies C494, C219, and JSB-1, and polyclonal antibody MDR Ab-1. There were no marked differences in optical densities of immunohistochemical staining near bile canaliculi of control and dieldrin-fed fish. Induction of xenobiotic binding capacity in cytosol of dieldrin-fed rainbow trout at least partially explained altered DMBA disposition in fish pretreated with this cyclodiene insecticide.

Hepatic microsomal enzyme activity in the koala and tammar wallaby: high 17β-hydroxysteroid oxidoreductase activity in koala liver microsomes

We have studied the hepatic microsomal xenobiotic metabolising capacity of koala ( Phascolarctos cinereus) and tammar wallaby ( Macropus eugenii). Total cytochrome P450 content in hepatic microsomes from koala (0.87±0.18 nmol/mg protein, n=4, mean (S.D.) and rat were comparable while tammar wallaby displayed reduced P450 content (0.24±0.04 nmol/mg protein). Associated microsomal activities (NADPH cytochrome P450 reductase, aminopyrine N-demethylation, aniline hydroxylation, and androstenedione 6β- and 16α-hydroxylation) in koala liver were similar to or reduced relative to rat. Hepatic microsomal NADPH-supported 17β-hydroxysteroid oxidoreductase (17β-HSOR) activity was significantly higher in koala (9.99±3.08 nmol/mg protein/min) than in tammar wallaby liver (0.86±0.16 nmol/mg protein/min). However, when NADH was utilised as cofactor the activity was similar in both marsupial species (koala, 1.44±0.84 nmol/mg protein/min; tammar wallaby, 1.52±0.44 nmol/mg protein/min). Michaelis–Menten parameters for the kinetics of 17β-HSOR androstenedione reduction by NADPH and NADH were determined in the koala. The K m for androstenedione was of the order of 1.9–4 μM ( n=4) irrespective of the cofactor used, whilst the K m for NADPH was 0.04–0.05 μM ( n=2) and for NADH was 134–430 μM ( n=2). Potential inhibitors were evaluated for their effects on NADPH-mediated 17β-HSOR activity with menadione and, to lesser extents, menthone, benzaldehyde and metyrapone eliciting significant inhibition. From detailed kinetic studies menthone was found to be an uncompetitive inhibitor of the activity in koala liver ( K i 220 μM).