Metabolism of nitrosamines by purified rabbit liver cytochrome P-450 isozymes

The metabolism of nitrosamines by microsomal cytochrome P-450 (P-450) isozymes was studied in a reconstituted monooxygenase system. P-450 LM2, LM3a, LM3b and LM3c, LM4, and LM6 were purified, respectively, from the livers of phenobarbital-treated, ethanol-treated, untreated, isosafrole-treated, and imidazole-treated rabbits. Of these isozymes, LM3a had the highest N-nitrosodimethylamine demethylase (NDMAd) activity with a Km of 2.9 mM and Vmax of 9.3 nmol/min/nmol. LM2, LM4, and LM6 exhibited NDMAd activity only at high N-nitrosodimethylamine concentrations, and isozymes LM3b and LM3c had poor activity even at the highest substrate concentrations examined. LM2, however, was more active than LM3a in the metabolism of N-nitrosomethylaniline. With each isozyme (LM3a or LM4), only one Km for NDMAd was observed, whereas with rabbit liver microsomes, multiple Km of 0.07, 0.27, and 36.8 mM were obtained. P-450 isozymes also catalyzed the denitrosation of nitrosamines at rates comparable to or lower than the demethylation, and the ratio of these two reactions was different with different nitrosamines. 2-Phenylethylamine and 3-amino-1,2,4-triazole, which were believed previously to affect NDMAd by mechanisms independent of P-450, were shown to be potent inhibitors of P-450-dependent NDMAd. These results further establish the role of P-450 isozymes in the metabolism of nitrosamines and indicate that LM3a is apparently responsible for the increased N-nitrosodimethylamine metabolism associated with ethanol treatment.     

Effect of diallyl sulfide on rat liver microsomal nitrosamine metabolism and other monooxygenase activities

It has been reported that p.o. administration of diallyl sulfide (DAS), a naturally occurring component of garlic (Allium sativum), inhibits 1,2-dimethylhydrazine-induced colon and liver cancer in rodents. A possible mechanism for this protective effect is inhibition of hepatic activation of the procarcinogen. The effect of DAS on P450IIE1, an isozyme of cytochrome P-450 which is active in the oxidative metabolism of dimethylhydrazine, was conveniently assayed in the present study by determination of N-dimethylnitrosamine demethylase (NDMAd) activity at 1 mM N-dimethylnitrosamine in Sprague-Dawley rat liver microsomal incubations. DAS was found to be a competitive inhibitor of NDMAd, in contrast to the irreversible inactivation of NDMAd produced by carbon tetrachloride incubated under similar conditions. The inhibition by DAS of the demethylation of several substrates was selective. The thioether was most potent against N-dimethylnitrosamine, less effective against N-nitrosomethylbenzylamine, and essentially ineffective against benzphetamine and ethylmorphine. Microsomes prepared at 3 h after DAS administration (200 mg/kg in corn oil intragastrically) showed moderate inhibition (less than 30% inhibition compared to control microsomes) of several demethylase activities; however, microsomes prepared 18 h posttreatment showed a marked decrease (about 80% inhibition compared to controls) in NDMAd activity, minor effects on other demethylase activities, and a 6-fold increase in pentoxyresorufin dealkylation. These trends at 18 h agreed with immunoblot analyses which showed suppression in the level of P450IIE1 and an elevation in P450IIB1. The selective inhibition of P450IIE1 activity and suppression of its level in microsomes may contribute to the reported chemoprotective effects of DAS.     

Metabolism and activation of N-nitrosodimethylamine by hamster and rat microsomes: comparative study with weanling and adult animals

It has been reported that hamster liver preparations are more effective for the metabolic activation of N-nitrosodimethylamine (NDMA) to a mutagen than rat liver preparations. The enzymatic basis for this phenomenon, however, has not been clearly elucidated. The present study was undertaken to examine the enzymology of NDMA metabolism by different hepatic subcellular fractions prepared from hamsters and rats of two different ages, and to investigate the correlation between the metabolism and the activation of NDMA to a mutagen for Chinese hamster V79 cells. The content of cytochrome P-450 was approximately 1.5-fold higher in hamster microsomes than in rat microsomes from both ages (1.19-1.38 versus 0.73-0.83 nmol P-450/mg protein). Weanling hamster microsomes exhibited multiple apparent Km values for NDMA metabolism as did weanling rat microsomes. The apparent Km I value of NDMA demethylase (NDMAd) in hamster microsomes was about one-half that in rat microsomes (36 versus 83 microM) with corresponding Vmax values of 2.09 and 2.57 nmol/min/nmol P-450. The Km I values for denitrosation did not differ from the corresponding values for NDMAd with Vmax values of 0.17 and 0.22 nmol/min/nmol P-450 for hamster and rat microsomes, respectively. These apparent Km values were affected neither by sonication nor by the presence of cytosolic proteins in S9 fractions. Adult rat liver microsomes showed less than one-half the NDMAd activity in weanling rat liver microsomes, whereas such age difference was not observed in hamster liver microsomes. This result was confirmed by Western blotting showing the levels of P-450ac (an acetone-inducible form of P-450) of these microsomes at comparable levels to their NDMAd activities. NDMAd was highly correlated to the metabolic activation of NDMA to a mutagen for V79 cells in an activation system mediated by microsomes prepared from hamsters and rats of different ages. The results from this study clearly demonstrate the enzymatic basis for the more effective metabolism of NDMA in adult hamsters than in adult rats.     

In-vivo and in-vitro evaluations for the effect of cyclophosphamide on the carcinogen metabolizing system of mouse-liver microsomes

The effect of the antineoplastic immunosuppressive alkylating agent cyclophosphamide (CPhA) on the modification of the carcinogen-metabolizing capacity was studied in vivo in mouse liver microsomes at different durations of treatment, from one to six consecutive days. The in vitro effect of increasing concentrations of the drug upon this enzyme system was also investigated. Following the administration of CPhA, a significant time-dependent decrease was observed in the activity of the low substrate level of the hepatic microsomal N-nitrosodimethylamine demethylase (NDMAdII). The high substrate level of the enzyme (NDMAdII) also exhibited a similar decrease which was not a subject for the treatment intervals where the greatest decrease (-60%; p<0.05) emerged at day 3 of the administration-point. The activity of the aryl hydrocarbon (benzo(alpha)pyrene) hydroxylase (AHH) revealed a significant increase at the single dose of CPhA, while at the repeated dose treatment (for 3 days) no alteration was noticed in the enzyme activity. This figure of expression in AHH was reversed to a significant inhibition at the 6 day-repeated dose, the time-point at which an almost identical effect was also observed in the hepatic content of cytochrome P450. The alterations in the metabolism of NDMA and benzo(alpha)pyrene which had been seen in the in vivo assays was further confirmed by the results of the in vitro experiment.     

Alteration in the capacities of carcinogen metabolizing system of mouse-livers during pretreatment with various antineoplastic agents

The effect of treatment with antineoplastic drugs on the modification of the carcinog en-metabolizing capacity was studied in mice liver microsomes at different durations as a single and as a repeated dose treatment. It is generally demonstrated that there is a commonality of influence for each specific antineoplastic group on the expression of the mixed function mono-oxygenases. The antineoplastic alkylating agents (chlorambucil, melphalan and busulfan) significantly increased the activity of carcinogen metabolizing enzymes in contrary to the effects observed for the antibiotic actinomycin-D. The antimetabolite agents (5-flourouracil and methotrexate), on the other hand, although decreased these activities when administered as a single dose, a pronounced increase was observed at the repeated dose treatment. Significant increases in the activity of N-nitrosodimethylamine demethylases I and II and aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase were observed with chlorambucil when administered as a single or repeated doses. Similar effects was observed when the animals were treated with repeated doses of melphalan, busulfan and 5-flourouracil. Contrary to these effects, inhibition in the enzyme activity was exhibited when actinomycin-D was administered for either single or repeated dose treatment. The hepatic content of cytochrome P450 was significantly increased with all the administered drugs, except busulfan, when applied repeatedly. The implication of such alterations in the capacities of carcinogen metabolizing enzymes for antitumour-induced toxicity and carcinogenicity are discussed.     

Enzyme kinetics of N-nitrosodimethylamine demethylase in rodents and humans

A variety of Km values have been reported for hepatic microsomal N-nitrosodimethylamine demethylase (NDMAd). We demonstrated previously that the biologically important, high affinity (KmI) form of microsomal NDMAd is manifested by cytochrome P450IIE1 (also known as P450ac and P450j). The KmI value of NDMAd was, however, affected greatly by assay conditions: the possible presence of inhibitors and the presence of cytochrome b5. We re-examined the KmI value by testing the effect of enzyme concentrations and of different types of enzyme preparations on the Km. The KmI value ranged from 15 to 22 microM, as estimated by the direct linear plot, using a microsomal protein concentration in the range of 0.1 to 0.8 mg/ml with correction for substrate utilization. A slight yet significant dependency of microsomal protein concentration on the Km (r = 0890; p less than 0.05) was seen. When five different microsomal preparations were compared, the KmI value ranged from 14 to 24 microM (median, 20 microM), as estimated by the direct linear plot. The Km estimated by the commonly used Eadie-Hofstee plot did not differ from that by the direct linear plot. These Km values are close to the values obtained in studies with isolated cells and tissue slices. The KmI form of NDMAd (P450IIE1 and its orthologues) is present in rats, mice, rabbits, hamsters and guinea-pigs. It is responsible for the age-dependent differences between rats and hamsters and for the sex-related differences in mouse kidneys, and for the bioactivation and toxicity of NDMA. This enzyme also exists in human liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phenethyl isothiocyanate, a carcinogenesis inhibitor, on xenobiotic-metabolizing enzymes and nitrosamine metabolism in rats.

Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, has been shown to inhibit chemical carcinogenesis, possibly due to its ability to block the activation or to enhance the detoxification of chemical carcinogens. The present study was conducted to elucidate the biochemical mechanisms involved by characterizing the effects of PEITC on phase I and phase II xenobiotic-metabolizing enzymes. A single dose of PEITC to F344 rats (1 mmol/kg) decreased the liver N-nitrosodimethylamine demethylase (NDMAd) activity (mainly due to P450 2E1) by 80% at 2 h and the activity of NDMAd remained decreased by 40% at 48 h after treatment. The liver pentoxyresorufin O-dealkylase (PROD) activity and P450 2B1 protein level were elevated 10- and 7-fold at 24 h after treatment respectively. The liver microsomal ethoxyresorufin O-dealkylase (EROD) (mainly due to P450 1A) and erythromycin N-demethylase (mainly due to P450 3A) activities were decreased at 2-12 h after treatment and recovered afterwards. The lung microsomal PROD and EROD activities were not significantly affected; whereas, the nasal microsomal PROD and EROD activities were decreased by 40-50%. After a treatment with PEITC, the rates of oxidative metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were decreased in liver microsomes by 40-60% at 2 h and recovered gradually; the rates in lung microsomes were markedly decreased by 60-70% at 2 h and remained at the decreased level at 24 h; and the rates in nasal mucosa microsomes were decreased gradually with the lowest activities observed at 18 h (50%) followed by a gradual recovery. Furthermore, the treatment with PEITC resulted in a maximal 5-fold increase of NAD(P)H:quinone oxidoreductase and 1.5-fold increase of glutathione S-transferase activities in the liver, but the activities of these two enzymes were not significantly affected in the lung and nasal mucosa. The sulfotransferase activity in the liver was decreased by 32-48% at 24-48 h after treatment; the nasal activity was increased by 1.8- to 2.5-fold, but the lung activity was not significantly changed. The hepatic UDP glucuronosyltransferase activity was slightly decreased at 2 h but slightly increased at 48 h after treatment, but no changes were observed for the lung and nasal activities. The study demonstrates that PEITC selectively affects xenobiotic-metabolizing enzymes in the liver, lung and nasal mucosa and it is especially effective in inhibiting the P450-dependent oxidation of NNK in the lung and of NDMA in the liver.     

Induction of microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450IVA1 (P-450LA omega) by dehydroepiandrosterone in rats: a possible peroxisomal proliferator

Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-450 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.4-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.45% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-450 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-450 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and 4 of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-450 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-450 reductase and at least one form of cytochrome P450 (P-450IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-450 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-450 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of androstenedione and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemomodulatory Effect of Moringa Oleifera,Lam, on Hepatic Carcinogen Metabolising Enzymes,Antioxidant Parameters and Skin Papillomagenesis in Mice

The modulatory effects of a hydro-alchoholic extract of drumsticks of Moringa oliefera Lam at doses of 125 mg/ ‍kg bodyweight and 250 mg/ kg body weight for 7 and 14 days, respectively, were investigated with reference to drug ‍metabolising Phase I (Cytochrome b5 and Cytochrome P450 ) and Phase II (Glutathione-S- transferase) enzymes, ‍anti-oxidant enzymes, glutathione content and lipid peroxidation in the liver of 6-8 week old female Swiss albino ‍mice. Further, the chemopreventive efficacy of the extract was evaluated in a two stage model of 7,12 – ‍dimethylbenz(a)anthracene induced skin papillomagenesis. Significant increase (p<0.05 to p<0.01) in the activities ‍of hepatic cytochrome b5, cytochrome P450, catalase, glutathione peroxidase ( GPx ), glutathione reductase (GR), acid ‍soluble sulfhydryl content (-SH ) and a significant decrease ( p<0.01 ) in the hepatic MDA level were observed at ‍both dose levels of treatment when compared with the control values. Glutathione-S- transferase ( GST )activity was ‍found to be significantly incr eased (p<0.01 ) only at the higher dose level. Butylated hydr oxyanisol (BHA ) fed at a ‍dose of 0.75% in the diet for 7 and 14 days (positive control ) caused a significant increase (p<0.05 to p<0.01) in the ‍levels of hepatic phase I and phase II enzymes, anti- oxidant enzymes, glutathione content and a decrease in lipid ‍peroxidation. The skin papillomagenesis studies demonstrated a significant decrease (p<0.05 ) in the percentage of ‍mice with papillomas, average number of papillomas per mouse and papillomas per papilloma bearing mouse when ‍the animals received a topical application of the extract at a dose of 5mg/ kg body weight in the peri-initiation phase ‍7 days before and 7 days after DMBA application, Group II ), promotional phase (from the day of croton oil application ‍and continued till the end of the experiment, Group III ) and both peri and post initiation stages (from 7 days prior ‍to DMBA application and continued till the end of the experiment, Group IV) compared to the control group (Group ‍I ). The percentage inhibition of tumor multiplicity has been recorded to be 27, 72, and 81 in Groups II, III, and IV, ‍respectively. These findings are suggestive of a possible chemopreventive potential of Moringa oliefera drumstick ‍extract against chemical carcinogenesis.     

Effect of chlorpromazine hydrochloride on carcinogen-metabolizing enzymes: liver microsomal dimethylnitrosamine demethylase, 4-dimethylaminoazobenzene reductase, and aryl hydrocarbon hydroxylase

Investigation of the effect of chlorpromazine hydrochloride (CPZ) on some enzymes involved in the metabolism of the carcinogens 4-dimethylaminoazobenzene (DAB), benzo[a]pyrene, and dimethylnitrosamine (DMN) revealed that CPZ inhibited hepatic microsomal DAB reductase, aryl hydrocarbon hydroxylase, and DMN demethylase II but markedly activated DMN demethylase I. Inhibition of these enzymes in vitro was proportional to the concentrations of CPZ. The effect of CPZ on DMN demethylase also depended on the concentrations of DMN and CPZ in the incubation mixture. Mechanisms to account for the inhibition of DAB reductase were suggested. Aryl hydrocarbon hydroxylase and DMN demethylase II activities of the 100,000 x g hepatic microsomal fraction were activated by 33 and 61%, respectively, over the control values after a single injection of CPZ into F344 rats, but no effect on DAB reductase and DMN demethylase I activities was observed. Microsomal concentrations of protein and cytochrome P450 were not appreciably altered by CPZ treatment, whereas the level of microsomal NADPH cytochrome c reductase was slightly increased over control values. A similar effect on the drug-metabolizing enzymes was found during pretreatment of rats with CPZ for 5 days, except that the NADPH cytochrome c reductase was increased by 33% over the control values.     

Effect of a Prudhoe Bay crude oil on hepatic and renal peroxisomal {beta}-oxidation and mixed-function oxidase activities in rats

The effect of oral administration of a Prudhoe Bay crude oil(PBCO) to male rats (PBCO, 2.6 g/kg body weight, daily) for5–12 days on hepatic and renal microsomal mono-oxygenaseactivities and peroxisomal ß-oxidation has been investigated.PBCO administration leads to liver enlargement. This is associatedwith induction of microsomal cytochrome P-450 levels (1.6- to2.0-fold) and dependent mixed-function oxidase activities (7-ethoxyresorufin-O-deethylaseand 7-pentoxyresorufin-O-depentylase, representing cytochromeP-450I and cytochrome P-450IIB isoenzymes respectively, 9-to15-fold; -oxidation of lauric acid representing the cytochromeP-450IVA1 isoenzyme, 1.4- to 1.5-fold) along with peroxisomalß-oxidation (palmitoyl CoA oxidation, 2- to 5-fold).It was observed that rats exposed to PBCO showed an increasein renal microsomal cytochrome P-450 content (1.6- to 2.3-fold),cytochrome P-450I activity (5- to 8-fold) and -oxidation activity(1.3- to 1.4-fold). However, renal peroxisomal ß-oxidationwas unaltered. Serum total triglycerides were lowered by 41–46%after PBCO exposure. These results suggest that induction ofperoxisomal ß-oxidation and possibly mono-oxygenasesmay be related to the carcinogenic/tumorigenic potential ofcrude oil.     

The induction and competitive inhibition of a high affinity microsomal nitrosodimethylamine demethylase by ethanol

The effects of ethanol on the metabolism of nitrosamines byrat liver microsomes have been studied. Treatment of rats with10 or 15% ethanol in drinking water for 3 days causes a 4- to5-fold enhancement in microsomal N-nitrosodimethylamine demethylase(NDMAd) activity and a 40–60% increase in gross P-450content. The enhancement is mainly due to the induction of alow K_m form (K_m = 0.07 mM) of NDMAd. The treatment induces proteinspecies with molecular weights between 50 000 and 52 000, someof which are believed to be P-450 isozymes with high affinityto NDMA. In addition to NDMA, treatment with ethanol also enhancesthe metabolism of N-nitroso-N-methylethylamine, N-nitrosomethylamline,and N-nitroso-N-methylbenzylamine. When added to the incubationmixture, ethanol and its homologs inhibit the demethylationof these nitrosamines by microsomes. Ethanol is a competitiveinhibitor of the low K_m NDMAd with a K_i of 0.31 mM and is lesseffective in inhibiting the metabolism of more lipophilic nitrosamines.     

3-Methylcholanthrene-inducible liver cytochrome(s) P450 in female Sprague-Dawley rats: possible link between P450 turnover and formation of DNA adducts and I-compounds

The hepatic cytochrome P450s are mixed-function oxidases whichmetabolize a wide variety of xenobiotics and endobiotics, andalso bioactivate carcinogens such as 3-methyl-cholanthrene (MC)to reactive metabolites capable of forming DNA adducts. To investigatepossible relationships between cytochrome P450 induction andcovalent DNA modifications (adducts and I-compounds), femaleSprague-Dawley rats were i.p. treated with MC (25 mg/kg) incorn oil (CO), once daily for 4 days. Controls received CO only.Animals were euthanized at 1, 8, 15, 28 and 45 days after thelast MC treatment, and liver microsomal cytochrome P450, ethoxy-coumarinO-deethylase (ECD) and ethoxyresorufin O-deethylase (EROD) activitieswere determined. Liver DNA adducts and I-compounds were analyzedby ³²P-postlabeling. A significant induction of the levels ofP450, ECD and EROD activities was noted in MC-treated rats,and elevated enzyme levels persisted for about 6 weeks aftercessation of MC administration. Linear decay of total P450,ECD and EROD activities as a function of time was observed.MC induced 11 DNA adducts in liver, which were resolved by thin-layerchromatography (TLC) and persisted at high levels throughoutthe study. On the other hand, MC elicited a significant depletionof both non-polar and polar I-compounds (age-dependent DNA modificationsdetectable by ³²P-postlabeling in rodent tissues without knownexposure to carcinogens). Level of most I-compounds returnedto normal at 45 days, and this paralleled the return of P450-relatedactivities to normal. These results suggest a possible linkbetween P450 turnover, DNA adduct formation, and I-compounddepletion.     

Induction of monooxygenases in rhesus monkeys by 3-methylcholanthrene: metabolism and mutagenic activation of N-2-acetylaminofluorene and benzo(a)pyrene.

Induction of content and activity of hepatic and lung cytochrome P450-dependent monooxygenases by 3-methylcholanthrene (MCA) was studied in male rhesus monkeys. Hepatic microsomes were concomitantly studied for mutagenic activation of N-2-acetylaminofluorene (AAF) as well as with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hepatic N-hydroxylation of AAF was increased sixfold to sevenfold after MCA treatment; aryl hydrocarbon hydroxylase was similarly increased in both liver and lung. The hepatic cytochrome P450 content was increased by 50%, and the Soret maximum in the CO-hemoprotein complex was shifted to the blue by 2 nm after MCA induction. Electrophoresis of hepatic microsomes from MCA-treated monkeys showed an increase in a polypeptide band of 54,000 molecular weight. Mutagenic activation of AAF by liver microsomes from untreated rhesus monkeys was low but was increased 40-fold after MCA treatment. No effect was observed on the mutagenic activation of N-hydroxy-2-acetylaminofluorene by liver microsomes after MCA treatment. Rhesus monkeys may provide a model for evaluation of the role of polycyclic hydrocarbon induction in chemically caused cancer and toxicity in primates.     

Induction and suppression of murine CYP-mediated biotransformation by dithianon: organ- and sex-related differences.

With the aim of evaluating the co-carcinogenic properties of dithianon, the regio- and stereo-selective hydroxylation of testosterone was used as a multibiomarker of effect for cytochrome P450 (CYP) changes. CYP-catalysed reactions have been studied in liver, kidney and lung microsomes from male and female Swiss albino CD1 mice treated i.p. with single (3 or 6 mg/kg body wt.) or repeated (3 mg/kg body wt. daily for 3 days) doses of this fungicide. Induction or suppression was recorded under various situations in different organs and sexes. In liver, all testosterone hydroxylase (TH) activities were increased in the single treatment from 2.8- (6beta-, 16alpha- and 16beta-TH activities) to 16-fold (2beta-TH activity) in males at the lower dose. In contrast, activities were reduced from 33.3% (16beta- and 17-TH activities, lower dose) to 66.4% (16beta-TH activity, higher dose) in females. In kidney, a similar pattern of modulation was achieved: induction from 2.9- to 5-fold (6beta- and 2alpha-TH activities, higher and lower doses, respectively) in males; suppression from 47.4 to 50.2% (2alpha- and 2beta-TH activities, either at lower or higher doses) in females. In lung, a significant induction ranging from 7.1- to 29.3-fold (16alpha- and 2alpha-TH activities, respectively, lower dose) in males, and up to a 7-fold increase (2beta-TH activity, higher dose) in females was obtained. After repeated treatment, hepatic 6beta-, 16beta-, 2alpha- and 2beta-TH activities were reduced up to approximately 60% in males, whereas no effect was seen in females. In extrahepatic tissues, a generalized increase of different THs was observed. The increase of 6beta-TH activity (CYP3A-linked), one of the most representative isoforms in humans, was sustained in liver and kidney by means of Western immunoblotting, using rabbit polyclonal antibodies anti CYP3A1/2. On the whole, a complex pattern of induction/suppression of CYP-dependent reactions was achieved depending on sex and tissue. The data are consistent with co-toxic, co-carcinogenic and promoting potentials of this fungicide and provide information of interest in evaluating the risk associated with human exposure.     

Differential induction of fetal mouse liver and lung cytochromes P-450 by beta-naphthoflavone and 3-methylcholanthrene

Previous studies have shown that the incidences of liver and lung tumors in mice exposed transplacentally to 3-methyl-cholanthrene (MC) were significantly influenced by the sensitivity of both mothers and fetuses to induction of cytochrome(s) P-450 by polycyclic aromatic hydrocarbons. In order to delineate further the biochemical and molecular processes underlying the observed biological effects, the inductive effect of MC and beta-naphthoflavone (beta NF) on cytochrome P-450 was determined at the biochemical and molecular levels. C57BL/6 females were mated with DBA/2 males and treated i.p. on day 17 of gestation with olive oil alone, 150 mg/kg of beta NF or different doses of MC. At various times after injection the mothers were sacrificed and the fetuses removed for biochemical and molecular studies. MC caused maximal induction of aryl hydrocarbon hydroxylase (AHH) activity by 8 h in both the liver and lung. beta NF caused nearly maximal induction of AHH activity by 8 h in the lung but had little effect on liver AHH activity at this time. Maximal induction with beta NF occurred by 24 h in both organs. Addition of monoclonal antibody 1-7-1, specific for the MC-inducible forms of cytochrome P-450 (P-450IA1 and A2), to the incubation mixtures resulted in a 55-70% inhibition of AHH activity in both lung and liver assays, regardless of the inducing agent used, while having no effect on AHH activity from oil-treated mice. RNA blot analysis carried out in parallel with enzyme assays demonstrated that the levels of enzyme activity correlated very well with the levels of steady-state RNAs. MC caused maximal induction of P-450IA1 RNA levels 4 h after injection in both organs and a biphasic secondary increase was observed in the lung. Maximal levels of P-450IA1 RNA were seen at 12-16 h following injection of beta NF. However, the ratio of P-450IA1 RNAs present at 16 versus 2 h in the beta NF-treated liver appeared greater than that in the lung. P-450IA2 was also induced in fetal liver and lung, but at low levels relative to P-450IA1. The results indicate that the increase in functional AHH activity was primarily due to induction of cytochrome P-450IA1. The differences in induction kinetics observed for cytochromes P-450IA1 and A2 suggest that these enzymes exhibit both tissue- and inducer-dependent specificity.     

Liposomal amphotericin B (AmBisome) application and hepatic microsomal enzyme function in the rat.

In the present study we investigated the influence of AmBisome, a lyophilized liposomal amphotericin B formulation on various hepatic cytochrome P450-dependent mixed function oxidases, antipyrine clearance and hepatic glucose-6-phosphatase activity in rats. Animals were treated intravenously for 6 days with AmBisome (15 mg kg-1 body weight). Subsequently, the enzyme activities and cytochrome P450 concentrations were measured ex vivo in hepatic microsomes. Following AmBisome the activity of the microsomal ethoxycoumarin-O-deethylase increased significantly from 333 +/- 77 pmol mg-1 to 459 +/- 125 pmol mg-1, whereas benzpyrenhydroxylase and glucose-6-phosphatase did not change compared with the controls. Accordingly, antipyrine clearance was not affected by AmBisome treatment. Microsomal cytochrome P450 concentrations as well as total microsomal protein concentrations were not changed following treatment with AmBisome and it did not affect either serum levels of liver transaminases or bilirubin. The results show that the application of a high AmBisome dose had no adverse effects on a variety of microsomal hepatic enzymes and the antipyrine clearance in rats. Thus, it seems likely that AmBisome does not seriously impair metabolic liver function.     

Frequent, moderate-dose cyclophosphamide administration improves the efficacy of cytochrome P-450/cytochrome P-450 reductase-based cancer gene therapy

Transduction of tumor cells with a cyclophosphamide (CPA)-activating cytochrome P-450 (P450) gene provides the capacity for localized prodrug activation and greatly sensitizes solid tumors to CPA treatment in vivo. The therapeutic impact of this P450-based cancer gene therapy strategy can be substantially enhanced by cotransduction of P450 reductase, a rate-limiting component of P450-dependent intratumoral CPA activation. The present study examined the possibility of further improving P450/P450 reductase-based gene therapy by using a novel schedule of CPA administration, involving repeated CPA injection every 6 days and previously shown to have an antiangiogenic component. 9L gliosarcoma cells transduced with the CPA-activating enzyme couple P450 2B6/P450 reductase and grown s.c. in immunodeficient severe combined immunodeficient (scid) mice were repeatedly challenged with 140 mg/kg CPA every 6 days. Full tumor regression leading to eradication of six of eight tumors was observed when the tumor size at the time of initial drug treatment was approximately 400 mm(3) (approximately 1.5% of body weight). Little or no overt toxicity of the repeated CPA treatment regimen was observed. The same CPA schedule was much less effective in inducing regression of 9L tumors that were not transduced with P450/P450 reductase. Repeated CPA treatment of mice bearing large, late-stage P450/P450 reductase-transduced tumors (approximately 9-16% of body weight) resulted in major (> or =95%) regression in 15 of 16 tumors, with tumor eradication observed in 2 cases. Although CPA resistance was found to emerge in the population of P450/P450 reductase-transduced tumors, this resistance primarily involved a loss of expression of the transduced P450 and/or P450 reductase gene, rather than development of intrinsic cellular resistance to the activated form of CPA. These findings demonstrate that repeated CPA treatment on a 6 day schedule can be highly effective when combined with P450/P450 reductase gene therapy and suggest that repeated transduction of tumors with prodrug-activation genes may be necessary to achieve tumor eradication and a sustained therapeutic response.