Selective colocalization of lipid peroxidation and protein thiol loss in chemically induced hepatic preneoplastic lesions: the role of γ-glutamyltranspeptidase activity

A number of studies indicate that cell proliferation can be modulated by changes in the redox balance of (soluble and protein) cellular thiols. Free radical processes, including lipid peroxidation (LPO), can affect such a balance, and a role for LPO in multistage carcinogenesis has been envisaged. The present study was aimed to assess the relationships between the protein thiol redox status and the LPO process in chemically induced preneoplastic tissue. The Solt-Farber's initiation-promotion model of chemical carcinogenesis in the rat liver was used. In fresh cryostat sections, preneoplastic lesions were identified by the reexpression of γ-glutamyltranspeptidase (GGT) activity. In serial sections, different classes of protein thiols were stained; in additional sections, LPO was elicited by various prooxidant mixtures and determined thereafter by the hydroxynaphthoic hydrazide-Fast Blue B procedure. The incubation of sections in the presence of chelated iron plus substrates for GGT activity leads to the development of LPO in selected section areas closely corresponding to GGT-positive lesions, indicating the ability of GGT activity to initiate LPO. Protein-reactive thiols, as well as total protein sulfur, were decreased by 20–25% in cells belonging to GGT-positive preneoplastic nodules, suggesting the occurrence of oxidative conditions in vivo. The incubation of additional adjacent sections with the prooxidant mixture H₂O₂ plus iron(II), in order to induce the complete oxidation of lipid present in the section, showed a decreased basal concentration of oxidizable lipid substrate in GGT-rich areas. The decreased levels of both protein thiols and lipid-oxidizable substrate in GGT-positive nodules suggest that the observed GGT-dependent path-way of LPO initiation can be chronically operative in vivo during early stages of chemical carcinogenesis, in cells expressing GGT as part of their transformed phenotype.     

Antiproliferative and antioxidant potential of beta-ionone against benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice

Nowadays, in developing countries like India, incidence of lung cancer is increasing rapidly, and as a consequence it has become the most common cause of malignancy-associated death. This study is aimed to evaluate the therapeutic efficacy of beta-ionone (ION), a precursor for carotenoids against benzo(a)pyrene [B(a)P]-induced lung carcinogenesis. B(a)P (50 mg/kg body weight, orally twice a week for 4 successive weeks)-induced lung cancer in mice was assessed both in tissue and serum in terms of increase LPO and tissue marker enzymes, such as aryl hydrocarbon hydroxylase, γ-glutamyl transpeptidase, 5'-nucleotidase, and lactate dehydrogenase, and serum tumor markers such as carcinoembryonic antigen and neuron-specific enolase with concordant decrease in activities of tissue enzymic and non-enzymic antioxidants were observed on the treatment of ION (60 mg/kg body weight, orally twice a week for 16 weeks) significantly attenuated LPO and restored all cancer marker enzymes and antioxidants levels to near normal, which indicates the anticancer effect of ION. This was further confirmed by histological staining of argyrophilic nucleolar organizer region and histopathological analysis of lung tissue, immunohistochemical and immunoblot analysis of proliferating cell nuclear antigen. Overall findings suggested that the ION effectively ameliorated the lung carcinogenesis, which is attributed to the antiproliferative and antioxidant potential through free radical scavenging property.     

Lycopene modulates initiation of N-nitrosodiethylamine induced hepatocarcinogenesis: Studies on chromosomal abnormalities,membrane fluidity and antioxidant defense system

Oxidative damage due to free radicals generated during nitrosamine metabolism has been suggested as one of the major cause for the initiation of hepatocarcinogenesis. Lycopene, is a well known antioxidant and have promising preventive potentials, however the mechanism of action remain hypothetical and unclear. To investigate the involvement of lycopene extracted from tomatoes (LycT) against oxidative stress induced deleterious effect of N-nitrosodiethylamine (NDEA) on cellular macromolecules, female Balb/c mice were divided in four groups: Control, NDEA (cumulative dose of 200 mg NDEA/kg body weight injected intraperitoneally in 8 weeks), LycT (5 mg/kg body weight given orally on alternate days, throughout the study) and LycT + NDEA (co-administration of LycT and NDEA). NDEA treatment commenced after 2 weeks of LycT administration. At the end of NDEA exposure i.e., at 10th week, enhanced activities of hepatic phase I enzymes, levels of reactive oxygen species (ROS), lipid peroxidation (LPO) was observed in NDEA group which may have contributed in chromosomal aberrations, enhanced micronucleated cell score, membrane fluidity and serum liver marker enzymes. A significant decrease in enzymatic and non-enzymatic antioxidant system could delineate the mechanism behind such NDEA insults. LycT pre-treatment to NDEA challenged group showed lower chromosomal abnormalities, micronucleated cells score, ROS, LPO levels and liver enzymes. Lycopene aids in normalizing the membrane fluidity and enhancing the activity of antioxidant enzymes and reduced glutathione which could account for the reduced oxidative damage in LycT + NDEA group. It seemed that lycopene supplementation target multiple dys-regulated pathways during initiation of carcinogenesis. Thus, dietary supplementation with lycopene can serve as an alternate measure to intervene the initiation of carcinogenesis.     

Inhibitory effects of Azadirachta indica on DMBA-induced skin carcinogenesis in Balb/c mice

Male Balb/c mice were divided into four groups on the basis of their respective treatments wherein mice of Group I served as controls. For induction of skin tumors, mice of Group II and IV were injected sub-cutaneously with 7,12-dimethylbenz(a)anthracene (DMBA). Mice of Group III and IV were administered aqueous Azadirachta indica leaf extract (AAILE) thrice a week throughout the experiment. After 14 weeks of the first DMBA injection, Group II and IV mice developed tumors. In the tumor-bearing mice that received AAILE (Group IV), a significant reduction in mean tumor burden and tumor volume was observed. The tumors were confirmed to be papillomas and interestingly, the extent of hyper-chromatia was observed to be much more in skin tumors of Group II mice vis a vis the mice receiving AAILE. An increase in the extent of lipid peroxidation was observed in tumorous tissue of Group IV when compared to that of Group II mice. Glutathione (GSH) content and the activities of GSH-based antioxidant enzymes viz. glutathione peroxidase (GPx) and glutathione reductase (GR) increased significantly in the skin tissues of all the groups of mice when compared to control counterparts. Catalase activity was found to decrease significantly in the skin of mice, which received AAILE treatment only (Group III). Activity of super-oxide dismutase (SOD) decreased significantly in all the tumorous tissues (Group II and IV mice). In light of the above observations, the role of AAILE in inhibition of DMBA-induced skin carcinogenesis is discussed in the present study.     

Modulatory influence of Brassica compestris Linn var sarson on phase-II carcinogen metabolizing enzymes and glutathione levels in mice

The present study reports the modulatory influence of 95% ethanolic extract from the seeds of B. compestris on the activity of phase-II enzymes such as glutathione S-transferase (GST), DT-diaphorase (DTD) and reduced glutathione (GSH) level in the skin, lung, kidney and forestomach of the mouse. Oral treatment with the seed extract at 800 mg/kg body wt. for 15 days significantly elevated GST in lung and forestomach and DT-diaphorase in forestomach and skin and GSH level in lung, kidney forestomach and skin. The lower dose 400 mg/kg body wt was effective only in inducing GST and DT-diaphorase activity in forestomach and reduced glutathione level in lung. The findings suggest that B. compestris seed extract may block or suppress the events associated with chemical carcinogenesis at least in part, by inducing metabolic detoxification of the carcinogen.     

Hepatoprotective activity of bacoside A against <Emphasis Type="Italic">N</Emphasis>-nitrosodiethylamine-induced liver toxicity in adult rats

N-Nitrosodiethylamine (DEN) is a notorious carcinogen, present in many environmental factors. DEN induces oxidative stress and cellular injury due to enhanced generation of reactive oxygen species; free radical scavengers protect the membranes from DEN-induced damage. The present study was designed to evaluate the protective effect of bacoside A (the active principle isolated from Bacopa monniera Linn.) on carcinogen-induced damage in rat liver. Adult male albino rats were pretreated with 15 mg/kg body weight/day of bacoside A orally (for 14 days) and then intoxicated with single necrogenic dose of N-nitrosodiethylamine (200 mg/kg bodyweight, intraperitonially) and maintained for 7 days. The liver weight, lipid peroxidation (LPO), and activity of serum marker enzymes (aspartate transaminases, alanine transaminases, lactate dehydrogenase, alkaline phosphatase, and γ-glutamyl transpeptidase) were markedly increased in carcinogen-administered rats, whereas the activities of marker enzymes were near normal in bacoside A-pretreated rats. Activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutatione-S-transferase, and reduced glutathione) in liver also decreased in carcinogen-administered rats, which were significantly elevated in bacoside A-pretreated rats. It is concluded that pretreatment of bacoside A prevents the elevation of LPO and activity of serum marker enzymes and maintains the antioxidant system and thus protects the rats from DEN-induced hepatotoxicity.     

Mechanisms of the anticancer effects of plant polyphenols. I. Blockade of initiation of carcinogenesis

Mechanisms of the anticancer effects of polyphenols, found in fruits, vegetables, spices and representing parts of daily nutrition, have been considered. These compounds may be the basis for the development of cancer preventive preparations. They can block initiation of carcinogenesis by inactivating exogenous or endogenous genotoxic molecules including reactive oxygen species (ROS). The other mechanism underlying polyphenol effects consists in inhibition of activity and synthesis of carcinogen-metabolizing enzymes. Plant polyphenols can induce expression of genes encoding antioxidant and detoxification enzymes and this also prevents initiation of carcinogenesis.     

Anticancer effect of ursolic acid stearoyl glucoside in chemically induced hepatocellular carcinoma

Hepatocellular carcinoma is one of the leading causes of death in cancer and yet no drug has proven to be a successful candidate for its treatment in advanced stages. Ursolic acid stearoyl glucoside (UASG) is a newly discovered triterpene in Lantana camara and there lies a possibility that it possess anti-hepatocellular carcinoma property. In the present study, we induced hepatocellular carcinoma in Wistar rats by diethylnitrosamine (DENA) and treated it with ursolic acid stearoyl glucoside. The ability to treat hepatocellular carcinoma was measured by comparing biochemical serum markers such as serum alanine aminotransferase, serum aspartate aminotransferase, serum alkaline phosphatase, and the specific marker for hepatocellular carcinoma, alpha fetoprotein. The histological studies of the livers were also performed. The results have shown significant elevated levels of these parameters as compared to normal control and the drug receiving groups have shown significant reduction in these marker levels. Histopathological studies also indicated the reduced liver damage in drug-treated groups. It was noted that a significant and dose-dependent reversal of DENA-diminished activity of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase, glutathione transferase, and the reduced DENA-elevated level of lipid peroxidation (LPO) with a marked change. UASG significantly suppressed free radical formation by scavenging the hydroxyl radicals. It also modulates the levels of LPO and markedly increases the endogenous antioxidant enzymes level in DENA-induced hepatocellular carcinogenesis.     

Evaluation of possible mechanisms of protective role of Tamarix gallica against DEN initiated and 2-AAF promoted hepatocarcinogenesis in male Wistar rats

We have previously reported that Tamarix gallica caused a marked inhibition of thioacetamide-induced hepatotoxicity, oxidative damage and early tumor promotion related events in the liver. These results strongly indicates that T. gallica may have chemopreventive potential. Therefore, in the present study, we examined the inhibitory effects of T. gallica methanolic extract on diethylnitrosamine (DEN) initiated and 2-acetyl aminofluorene (2-AAF) promoted liver carcinogenesis in male Wistar rats. Interestingly, it was found that T. gallica (25 and 50 mg/kg body wt.) resulted in a marked reduction of the incidence of liver tumors. The study was further histologically confirmed. Furthermore to understand the underlying mechanisms of chemopreventive action by T. gallica we evaluated the levels activities of hepatic antioxidant defense enzymes, ornithine decarboxylase activity and hepatic DNA synthesis as a marker for tumor promotion since direct correlation between these marker parameters and carcinogenicity have been well documented. Treatment of male Wistar rats for five consecutive days with 2-AAF i.p. induced significant hepatic toxicity, oxidative stress and hyperproliferation. Pretreatment of T. gallica extract (25 and 50 mg/kg body wt.) prevented oxidative stress by restoring the levels of antioxidant enzymes and also prevented toxicity at both the doses. The promotion parameters induced (ornithine decarboxylase activity and DNA synthesis) by 2-AAF administration in diet with partial hepatectomy (PH) were also significantly suppressed dose-dependently by T. gallica. Therefore, we can conclude that ultimately the protection against liver carcinogenesis by T. gallica methanolic extract might be mediated by multiple actions, which include restoration of cellular antioxidant enzymes, detoxifying enzymes, ODC activity and DNA synthesis.     

Update on the effects of vitamins A, C, and E and selenium on carcinogenesis

The effects of vitamins A, C, and E and of selenium on carcinogenesis are briefly summarized and updated. These vitamins and minerals were selected because they have been studied extensively in recent years with a variety of carcinogenesis models. The consumption of vitamin A and its precursors (carotenoids) has been negatively correlated with cancer at a number of sites, particularly the lung. Animal investigations on vitamin A involvement in carcinogenesis have generally been of three types: those assessing the effect of vitamin A deficiency, the effect of excess vitamin A, or the effect of supplementation with synthetic analogs of vitamin A. Vitamin A deficiency had no effect on salivary gland carcinogenesis, enhanced urinary bladder, lung, and liver carcinogenesis, and inhibited colon carcinogenesis. Excess of various forms of vitamin A enhanced or inhibited skin tumorigenesis, inhibited mammary carcinogenesis in rats (but not in mice), and carcinogenesis of the forestomach, liver, and urinary bladder (with one model, but not with another), or enhanced or did not influence lung carcinogenesis. Vitamin A analogs have enhanced or inhibited skin tumorigenesis, inhibited salivary gland, mammary, and urinary bladder carcinogenesis, enhanced tracheal and liver carcinogenesis, and either enhanced or inhibited pancreas carcinogenesis, depending upon the model employed. Although retinoids have been shown to inhibit carcinogenesis at many sites, numerous negative studies have been reported and some reports have indicated enhanced carcinogenesis. The most convincing evidence for the involvement of vitamin C in cancer prevention is the ability of ascorbic acid to prevent formation of nitrosamine and of other N-nitroso compounds. In addition vitamin C supplementation was shown to inhibit skin, nose, tracheal, lung, and kidney carcinogenesis, to either not influence or enhance skin, mammary gland, and colon carcinogenesis, and to enhance urinary bladder carcinogenesis, when given as sodium ascorbate, but not when given as ascorbic acid. Like vitamin C, vitamin E can inhibit nitrosation. Vitamin E was shown to inhibit skin, cheek pouch, and forestomach carcinogenesis, to enhance or inhibit colon carcinogenesis, and to have no effect on or to inhibit mammary gland carcinogenesis, depending upon the method of vitamin E administration or the level of dietary selenium or dietary fat. Selenium effects on carcinogenesis have been recently reviewed and the present discussion only updates this area by indicating that enhancement of carcinogenesis by dietary selenium supplements has been observed in the liver, pancreas, and skin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hepatoprotective potential of new steroid against carbon tetrachloride-induced hepatic injury

The present study was designed to evaluate the hepatoprotective effects of newly isolated stigmast-4, 20 (21), 23-trien-3-one (STO) against carbon tetrachloride-induced hepatic injury in Wistar albino rats. Hepatotoxicity was induced by the administration of a single intraperitoneal dose of CCl₄ (0.5 mL/kg CCl₄ in olive oil) in experimental rats. Three different doses (2.5, 5.0, and 10 mg/kg, p.o) of STO was administered to the test groups during whole experimental protocol. Changes in the activity of serum ALT, AST, ALP, TB, and TP, anti-oxidant enzymes like SOD, CAT, GPx, GST, and LPO were studied in CCl₄-induced hepatocellular carcinogenesis. The altered levels of serum ALT, AST, ALP, TB, and TP restored toward normalization significantly by STO in a dose dependant manner. The biochemical observations were supplemented with histopathological examination of rat liver sections. Meanwhile, it also produced a significant and dose-dependent reversal of CCl₄-diminished activity of anti-oxidant enzymes like SOD, CAT, GPx, GST, and the reduced CCl₄-elevated level of LPO. STO significantly prevented the increased levels of serum markers, also suppressed the free radical processes by scavenging hydroxyl radicals. It also modulates the levels of LPO and markedly increases the endogenous anti-oxidant enzymes level in CCl₄-induced hepatic injury.     

Effect of excessive intake of ascorbic acid on hepatic and extra-hepatic phase I and phase II drug metabolism in rat

Guinea pig is the animal model of choice for studies on effects of ascorbic acid (AA). However, rat is one of the largely used animals for investigations related to chemical carcinogenesis. Therefore, the present study was designed to evaluate the changes induced by high intake of the vitamin in xenobiotic and carcinogen metabolizing status of the organs. Male Wistar rats, dosed daily with 50 mg AA/100 g body weight for 10 weeks, demonstrated a small non-significant increase in hepatic, pulmonary and colon cytochrome P-450 (Cyt. P-450) contents, which was accompanied with a significant increase in hepatic and pulmonary arylhydrocarbon hydroxylase (AHH) activities. Phase II enzymes of drug metabolism responded in different ways to increased intake of AA. UDP-glucuronyltransferase (UDPGT) activity was unaffected in liver and colon, but it was increased (p less than 0.005) in lung. Activities of glutathione S-transferase (GST) were decreased in the three organs. Inducibility of AHH by 3-methylcholanthrene (MCA) or phenobarbital (PB) was largely reduced due to AA feeding. Besides this, MCA and PB had differential effects on enzymatic levels in AA fed rats. When compared with our earlier observations in guinea pig, it was found that rat responded similarly to guinea pig to increased intake of AA with regard to hepatic AHH, Cyt. P-450, UDPGT and GST, pulmonary AHH, Cyt. P-450 and Cyt. b5, and all studied colon enzymes, except GST.     

Role of lupeol and lupeol linoleate on lipemic-oxidative stress in experimental hypercholesterolemia

Epidemiological studies have shown that there is a positive correlation between the incidence of coronary heart disease (CHD) and the blood cholesterol level. To study the effect of plant derived triterpene, lupeol and its ester lupeol linoleate, on blood lipid status and oxidant stress in heart and hemolysate, male albino Wistar rats were fed high cholesterol diet (normal rat chow supplemented with 4% cholesterol and 1% cholic acid; HCD) for 30 days. A significant increase (p<0.05) in plasma total cholesterol (4.22 fold) and triglycerides (1.7 fold) was observed in HCD fed rats, along with elevated LDL (3.56 fold) and VLDL (1.99 fold) cholesterol and decreased HDL cholesterol (34.14%). Treatment with lupeol and its derivative normalized the lipid profile. The significant increase (p<0.05) in lipid peroxidation (LPO) was paralleled by significantly diminished (p<0.05) activities of antioxidant enzymes (SOD, CAT and GPx) and decreased (p<0.05) concentration of antioxidant molecules (GSH, Vit C and Vit E) in cardiac tissue and hemolysate of HCD fed rats. The oxidative tissue injury in hypercholesterolemic rats was substantiated by the increase in cardiac marker, serum CPK and the drop in its activity in the heart tissue. Lupeol and lupeol linoleate treatment decreased the LPO levels and increased enzymatic and nonenzymatic antioxidants. CPK activity in the treated group was comparable with that of the control. These observations highlight the beneficial effects of the triterpene, lupeol and its linoleate ester derivative, in ameliorating the lipidemic-oxidative abnormalities in the early stage of hypercholesterolemic atherosclerosis.     

Possible involvement of NO-mediated oxidative stress in induction of rat forestomach damage and cell proliferation by combined treatment with catechol and sodium nitrite

To clarify the mechanisms underlying forestomach carcinogenesis in rats by co-treatment with catechol and sodium nitrite (NaNO2), we investigated the involvement of oxidative stress resulting from reaction of the two compounds. Since generation of semiquinone radical, hydroxyl radical (*OH), and peroxynitrite (ONOO-) arose through the reaction of catechol with NO, we proposed that superoxide resulting from catechol oxidation reacted with excess NO, consequently yielding *OH via ONOO-. Male F344 rats were co-treated with 0.2% catechol in the diet and 0.8% NaNO2 in the drinking water for 2 weeks. Prior to occurrence of histological evidence indicating epithelial injury and hyperplasia, 8-hydroxydeoxyguanosine levels in forestomach epithelium significantly increased from 12 h together with appearance of immunohistochemically nitrotyrosine-positive epithelial cells. There were no remarkable changes in rats given each chemical alone. We conclude that oxidative stress due to NO plays an important role in induction of forestomach epithelial damage, cell proliferation, and thus presumably forestomach carcinogenesis.     

Effect of Cassia fistula Linn. leaf extract on diethylnitrosamine induced hepatic injury in rats

The hepatoprotective and antioxidant effect of Cassia fistula Linn. leaf extract on liver injury induced by diethylnitrosamine (DEN) was investigated. Wistar rats weighing 200+/-10g were administered a single dose of DEN (200mg/kg b.w., i.p.) and left for 30 days. For hepatoprotective studies, ethanolic leaf extract (ELE) of C. fistula Linn. (500mg/kg b.w., p.o.) was administered daily for 30 days. AST, ALT, ALP, LDH, gamma-GT and bilirubin were estimated in serum and liver tissue. Lipid peroxidation (LPO), SOD and CAT were also estimated in liver tissue as markers of oxidative stress. DEN induced hepatotoxicity in all the treated animals were evident by elevated serum ALT, AST, ALP and bilirubin levels and a simultaneous fall in their levels in the liver tissue after 30 days. Induction of oxidative stress in the liver was evidenced by increased LPO and fall in the activities of SOD and CAT. ELE administration for 30 days prevented the DEN induced hepatic injury and oxidative stress. In conclusion, it was observed that ELE of C. fistula Linn. protects the liver against DEN induced hepatic injury in rats.     

Effects of recombinant human interferon alpha on aryl hydrocarbon hydroxylase activity in cultured human peripheral lymphocytes

Interferon and its inducers are known to depress drug biotransformation in vivo by decreasing the levels of cytochrome P-450 (P450) monooxygenase system in the liver. However, very little is known about the effects of interferon on P450 in extrahepatic tissues. In this study we investigated the effects of a recombinant human interferon-alpha (rhIFN-alpha) on aryl hydrocarbon hydroxylase (P450IAI) in cultured human peripheral lymphocytes (HPL). Non-induced and induced (3-methylcholanthrene) mitogen activated lymphocytes were used throughout the study. rhIFN-alpha maximally depressed AHH activity to approximately 58% of control after 24 hrs of incubation in both non-induced and induced lymphocytes. However, after 48 hrs of incubation with rhIFN-alpha, AHH activity had recovered to 86% of control in induced cells and 61% in non-induced cells. rhIFN-alpha had no significant effect on either NADH cytochrome c reductase activity or on viable lymphocyte cell count. This is the first demonstration that rhIFN-alpha can have a direct depressive effect on a P450 dependent monooxygenase system in HPL.     

Hepatoprotective effect of pyrroloquinoline quinone against alcoholic liver injury through activating Nrf2-mediated antioxidant and inhibiting TLR4-mediated inflammation responses

The present study was aimed at investigating the hepatoprotective effect of pyrroloquinoline quinone (PQQ) against acute alcoholic liver injury in mice. Acute alcoholic liver injury model was established in mice, and they were administrated with PQQ to investigate its hepatoprotective effect. Our results shows that PQQ can significantly ameliorate acute alcoholic liver injury by decreasing the hepatic marker enzymes, including serum alanine transaminase (ALT) and aspartate transaminase (AST), and increasing the levels of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the liver. And PQQ can also significantly reduce the content of hepatic triglyceride (TG) and malondialdehyde (MDA). Moreover, PQQ attenuated alcohol-induced oxidative damage by activating NF-E2-related factor 2 (Nrf2)-mediated signaling pathway, and inhibiting Toll-like receptor 4 (TLR4)-mediated nuclear factor-kappa B (NF-κB) signaling pathway. Our findings have elucidated the liver protection mechanism of PQQ, which would encourage the further exploitation of PQQ as a hepatoprotective functional food.     

Stereoselective and regioselective hydroxylation of warfarin and selective O-dealkylation of phenoxazone ethers in human placenta

The oxidative metabolism of warfarin and a series of phenoxazone ethers was studied in two groups of human placentas which exhibited high or low levels of aryl hydrocarbon hydroxylase (AHH). Warfarin metabolism was stereoselective (mean R/S = 2.48) for the R-enantiomer and regioselective for the 6- and 8- positions in the high AHH group whereas warfarin metabolism in the low AHH group displayed no significant overall stereoselectivity (mean R/S = 1.24) and was regioselective for the 7- position. The high AHH group metabolized the methyl, ethyl, propyl and butyl ethers of phenoxazone rapidly, while the low AHH group catalyzed their biotransformation at very low or negligible rates. Neither group detectably metabolized phenoxazone or pentyloxyphenoxazone whereas both groups metabolized benzyloxyphenoxazone at low but similar rates. Rates of warfarin R-6 and R-8 hydroxylation were highly correlated with metabolism of benzo(alpha)pyrene (r = 0.99) and the C1-C4 phenoxazone ethers (r greater than 0.87), but poorly correlated with metabolism of benzyloxyphenoxazone (r less than 0.50). These data support the use of warfarin and the phenoxazone ethers as sensitive biochemical probes for P-450 isozymes in human extrahepatic tissues. They indicate the presence of a multiplicity of xenobiotic metabolizing P-450's in placental tissue which has not been exposed to inducing agents that elevate AHH.     

Characterization of biotransformation enzyme activities in primary rat proximal tubular cells

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.