Eino Hietanen

Carcinogenic nitrosamines: Free radical aspects of their action

NDMA and other nitrosamines may be activated into DNA binding intermediates by a cytochrome P450-dependent formation of alpha-nitrosamino radicals or photochemically. Within the catalytic site of cytochrome P450, these radical intermediates either combine with HO. to form alpha-hydroxynitrosamines or decompose into nitric oxide and N-methylformaldimine. In the presence of phosphate, nutagenic alpha-phosphonooxy derivatives are formed from radicals generated chemically/photochemically. Studies on lipid peroxidation, in vivo and in vitro, have further suggested that radicals are formed as intermediates from N-nitrosodialkylamines. The level of nitrosamine-induced lipid peroxidation parallels hepatocarcinogenicity in rats. These data, although preliminary, provide further evidence that free radical damage and DNA alkylation are involved in carcinogenesis induced by nitrosamines.

Monoclonal antibody-directed characterization of cytochrome P450 isozymes responsible for toluene metabolism in rat liver

Monoclonal antibodies (MAbs) were used to study the contribution of cytochromes P450IA1/IA2, P450IIB1/IIB2, P450IIC11/IIC6 and P450IIE1 to toluene side-chain (benzyl alcohol, BA, formation) and ring (o- and p-cresol formation) oxidation in liver microsomes from fed, one-day fasted, and phenobarbital (PB)-, 3-methylcholanthrene (MC)- and ethanol-treated rats. All rats were fed synthetic liquid diets. MAb 1-7-1 against P450IA1/IA2 inhibited markedly o-cresol formation and slightly p-cresol formation but not BA formation only in microsomes from MC-treated rats. MAbs 2-66-3, 4-7-1 and 4-29-5 against P450IIB1/IIB2 strongly inhibited BA, o-cresol and p-cresol formation only in PB-induced microsomes. MAb 1-68-11 against P450IIC11/IIC6 inhibited BA formation at high toluene concentration in the following order: fed greater than fasted greater than ethanol = MC greater than PB, and ethanol greater than or equal to fed = fasted greater than MC greater than PB on the basis of the percentage and net amount inhibition, respectively. MAb 1-91-3 against P450IIE1 inhibited BA formation at low toluene concentration, but not at high concentration, in the following order: ethanol greater than fasted = fed greater than MC, and ethanol greater than fasted greater than fed greater than MC on the basis of percentage and net inhibition, respectively. MAbs 1-68-11 and 1-91-3 also inhibited p-cresol formation at high and low toluene concentrations, respectively. These results indicate that (i) both P450IIE1 and P450IIC11/IIC6 are constitutive isozymes mainly responsible for the formation of BA and p-cresol from toluene as low- and high-Km isozymes, respectively; (ii) P450IIE1, but not P450IIC11/IIC6, is induced by one-day fasting and ethanol treatment; (iii) both P450IIE1 and P450IIC11/IIC6 are decreased by PB and MC treatments; (iv) P450IIE1 is inhibited by high concentration of toluene; (v) P450IIB1/IIB2 can contribute to the formation of BA, o- and p-cresol from toluene, while P450IAI/IA2 preferentially contributes to the formation of o-cresol.

Monoclonal antibody-directed characterization of benzene, ethoxyresorufin and pentoxyresorufin metabolism in rat liver microsomes

The contribution of cytochrome P450IA, P450IIB, P450IICII and P450IIEI to the oxidative metabolism of benzene, 7-ethoxyresorufin and 7-pentoxyresorufin was investigated using monoclonal antibodies (MAb) in liver microsomes from fed, one-day fasted, phenobarbital (PB)-, 3-methylcholanthrene (MC)- and ethanol-treated rats. Overall catalytic activity varied with different pretreatments and thereby the contribution of different P450s. MAb 1-91-3 against P450IIE1 did not influence alkoxyresorufin dealkylation but inhibited benzene aromatic hydroxylase (BAH) in relation to its increasing inducibility as follows: MC, PB (less than or equal to 48%) less than fed (less than or equal to 59%) less than fasted (less than or equal to 70%) less than ethanol (less than or equal to 91%). MAbs 2-66-3, 4-7-1 and 4-29-5, all against P450IIB, had no effect on 7-ethoxyresorufin O-deethylase (EROD) but inhibited the activities of high-Km BAH (greater than or equal to 58%) and 7-pentoxyresorufin O-depentylase (PROD) (greater than or equal to 96%) in PB-treated microsomes. MAb 1-7-1 against P450IA inhibited EROD (79%), PROD (50%) and high-Km BAH (42%) activities in MC-microsomes. MAb 1-68-11 against P450IIC11 inhibited EROD, PROD and high-Km BAH activities. Thus, P450IIE1 contributed to benzene metabolism as a low-Km BAH but not to alkoxyresorufin metabolism. P450IIB was responsible besides for the major part of 7-pentoxyresorufin metabolism also, selectively, for benzene hydroxylation at high benzene concentrations. P450IA contributed primarily to 7-ethoxyresorufin metabolism and only slightly to PROD and high-Km BAH activities. P450IIC11 contributed slightly to high-Km BAH and to alkoxyresorufin metabolism.

Plasma and lipoprotein lipid peroxidation in humans on sunflower and rapessed oil diets

The effects of natural mixed diets on lipid peroxidation were investigated in humans. In the first study, 59 subjects were fed a rapeseed oil-based diet rich in monounsaturated fatty acids (MUFA) and a sunflower oil-based diet rich in polyunsaturated fatty acids (PUFA) in a cross-over manner for three and a half weeks. The lipid peroxidation products in plasma were determined by measuring conjugated dienes and malondialdehyde (MDA). In a second study, plasma thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides, and the susceptibility of very low density lipoprotein + low-density lipoprotein (LDL) toin vitro oxidation were measured from subjects fed similar MUFA and PUFA diets for six week diets. No significant differences in plasma MDA or conjugated diene concentrations were found after the rapeseed oil diet or the sunflower oil diet in Study 1. In the second study, a small but significant decrease (P<0.05) in both lipid hydroperoxides and TBARS was observed in the LDL fraction after the sunflower oil diet. Thein vitro oxidation gave opposite results, showing increased oxidation after the sunflower oil diet. Despite a high intake of α-tocopherol during the oil peroids, no increase in plasma α-tocopherol was noticed in either study. The results suggest that moderate changes in the fatty acid composition in the Western-type diet may be adequate to affect lipoprotein susceptibility to oxidationin vitro, but there is considerable disparity with some indices ofin vivo lipid peroxidation.

Inducibility of Mucosal Drug-Metabolizing Enzymes of Rats Fed on a Cholesterol-Rich Diet by Polychlorinated Biphenyl, 3-Methylcholanthrene and Phenobarbitone

The results show that a cholesterol-rich diet changes the composition of mucosal membranes. A high cholesterol diet increases mucosal cholesterol and phospholipid contents. Cholesterol enhanced mucosal NADPH cytochrome c reductase and aryl hydrocarbon hydroxylase activities as well as mucosal UDP glucuronosyltransferase activity. When phenobarbitone or Clophen A 50 or 60 were administered intraperitoneally to cholesterol-fed rats, the hydroxylation and glucuronidation activities decreased to a lower level. 3-Methylcholanthrene was, however, able to maintain or increase mucosal hydroxylative enzymes and UDP glucuronosyltransferase. These results indicate that the drug-metabolizing enzymes of the intestinal mucosa behave very differently from those in the liver. Diet apparently has a regulatory effect on the induction of drug-metabolizing enzymes because only a very potent inducer, 3-methylcholanthrene, was able to maintain and even induce mucosal drug-metabolizing enzymes in rats fed on a high cholesterol diet, possibly through changes in the microenvironment of enzymes caused by cholesterol.

Monoclonal antibody characterization of hepatic and extrahepatic cytochrome P-450 activities in rats treated with phenobarbital or methylcholanthrene and fed various cholesterol diets.

Monoclonal antibodies (MAb) against 3-methylcholanthrene (MC)- and phenobarbital (PB)-inducible forms of cytochrome P-450 isozyme were used to characterize changes in aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase (ECDE) activities modulated by dietary cholesterol. Rats were induced by MC or PB, and immunochemical inhibition of AHH and ECDE activities was studied as an indication of changes in cytochrome P-450 isozyme patterns. Feeding of a cholesterol-free diet markedly decreased enzyme activities both in liver and in small intestinal mucosa, and the highest activities were observed after feeding rats a high (2%)-cholesterol diet for one month. As a control, a normal pelleted diet (0.1% cholesterol) was used; in rats fed this diet, intermediate levels of monooxygenase activities were present. Although no diet-dependent change in total AHH and ECDE activities was observed in kidneys and lungs, diet apparently modulated isozyme composition in the lungs, as indicated by a change in the immunochemical inhibition pattern with MAb; no such shift was observed in the kidneys. In liver and intestine, in addition to changes in total activity, isozyme composition was also altered, as indicated by inhibition of the catalytic activities of cytochrome P-450 by MAb. Our data infer that dietary cholesterol can: (i) modulate total monooxygenase activities, especially in the intestine; (ii) change the cytochrome P-450 isozyme composition in liver and intestine; (iii) change isozyme composition without changing overall enzyme activity, e.g. in lungs; and (iv) have no effect in a tissue (e.g. kidney) that lacks constitutionally the P-450 isozyme responsive to cholesterol.

Elevated Lipid Peroxidation in Rats Induced by Dietary Lipids and N-Nitrosodimethylamine and its Inhibition by Indomethacin Monitored Via Ethane Exhalation

The effect of dietary lipids alone or in combination with an administered carcinogen, N-nitrosodimethylamine (NDMA), on whole body lipid peroxidation was studied in rats in vivo. Groups of rats were fed diets containing 2%, 12.5%, or 25% of either saturated or polyunsaturated fat. Lipid peroxidation in individual animals was determined by measuring the concentration of ethane in exhaled air. Increased ethane exhalation was found in rats when the amount of dietary fat was increased from 2% to 12.5%, but animals receiving 12.5% or 25% fat in the diet exhaled ethane at similar rates. Rats consuming polyunsaturated fat exhaled more ethane than those eating saturated fat. In all groups, NDMA administration drastically increased ethane exhalation. Indomethacin completely blocked the increase in ethane exhalation caused by dietary lipids.

Effect of administration route of 3-methylcholanthrene on the inducibility of intestinal drug-metabolizing enzymes.

The inducibility of the mucosal drug-metabolizing enzymes of rat small intestine was studied by administering 3-methylcholanthrene either intragastrically or intraperitoneally. The aryl hydrocarbon hydroxylase activity was 4.1 times higher after intragastric than after intraperitoneal administration of methylcholanthrene. The ethoxycoumarin-O-diethylase activity was 11 times and UDP-glucuronosyltransferase (with p-nitrophenol as substrate) activity was 3 times higher after intragastric administration than after intraperitoneal administration. The epoxide hydratase activity was, on the other hand, 38% lower after intragastric administration of 3-methylcholanthrene than after intraperitoneal administration. The results suggest that compounds entering the body intragastrically, e.e. in the diet, might have profound enzyme-specific effects on the intestinal metabolic rates of drugs.

Enhancement of hepatic drug biotransformation rate by polychlorinated biphenyls in rats fed cholesterol-rich diet.

A combined effect of cholesterol and polychlorinated biphenyls (PCBs) on the microsomal drug hydroxylation and glucuronidation in the liver of the rat was studied. PCBs, Clophen A-50 and A-60, having an average chlorination degree of 50 and 60% affect the structure of microsomal membranes. It was found that Clophen A-60 increased the binding of trypsin- and digitonin-sensitive proteins to the membranes. Also it was found that PCBs enhanced the phsopholipid content of microsomes. PCBs increased the activity of hepatic NADPH cytochrome c reductase about 1.5-fold. Aryl hydrocarbon hydroxylase activity doubled with Clophen A-50 and quadrupled with Clophen A-60. Hepatic UDPglucuronosyltransferase activity was doubled with both PCBs. The enhancement in hepatic aryl hydrocarbon hydroxylase and in UDPglucuronosyltransferase was found to be lower in the presence of high cholesterol level in the diet when compared to earlier results. This is supposed to be due to the membraneous effects of cholesterol.

Determination of human apolipoprotein C-II by electroimmunoassay. Studies on standardization and determination before and after physical training

1. Human VLDL and HDL were fractionated by sequential ultracentrifugation until free of contaminant plasma proteins. 2. Column chromatofocusing method was used to isolate apolipoprotein C-II from apoVLDL and apo HDL. C-apoprotein peak was rechromatofocused and the second peak was the apo C-II (pI 4.7, homogeneous band on SDS slab gel). 3. New Zealand white rabbits were immunized with apo C-II. Antiserum gave a single precipitate are of identity between whole serum, apoVLDL, apoHDL and apo C-II. 4. Apo C-II concentration was measured by electroimmunoassay method. During standardization 1% Triton X-100 improved the rocket shapes and contours. Total delipidation did not affect the assay system and so the antigenic determinants of apo C-II are all available to antiserum. The lowest concentration of apo C-II possible to determine with this method was 70 ng/sample well. 5. There was no difference between the apo C-II values before (39.8 +/- 7.1 mg/l, n = 19) and after (41.6 +/- 6.4 mg/l, n = 19) moderate physical training among normolipemic subjects. 6. Specific immunoprecipitation technique was also used to determine apo C-II content in standard pool serum.