NADPH-cytochrome-P-450 reductase promoted hydroxyl radical production by the iron(III)-ochratoxin A complex.

The Fe3+ complex of ochratoxin A has been shown to produce hydroxyl radicals in the presence of NADPH and NADPH-cytochrome-P-450 reductase. ESR spin-trapping experiments carried out in the presence of the hydroxyl radical scavenger ethanol and the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide) produced ESR spectra characteristic of the hydroxyl radial-derived carbon-centered DMPO-alkoxyl radical adduct. Thus hydroxyl radicals produced by the Fe3(+)-ochratoxin A complex in the presence of an enzymatic reductase may be be partly responsible for ochratoxin A toxicity.

Possible role of an iron-oxygen complex in 4(S)-4-hydroxyochratoxin a formation by rat liver microsomes.

Rat liver microsomes were examined for their ability to oxidize the mycotoxin ochratoxin A (OTA) to 4(R)-4-hydroxyochratoxin A [(R)-4-OH-OTA] and 4(S)-4-hydroxyochratoxin A [(S)-4-OH-OTA] and to induce OTA-dependent lipid peroxidation. Microsomes isolated from rats pretreated with pregnenolone-16 alpha-carbonitrile greatly induced both (R)-4-OH-OTA and (S)-4-OH-OTA formation whereas isoniazid pretreatment primarily induced (S)-4-OH-OTA. (R)-4-OH-OTA and (S)-4-OH-OTA formation showed significant differences with respect to pH optima, effect of antioxidants, and iron chelators. (R)-4-OH-OTA showed a pH optimum of 6.5 and was not inhibited by the antioxidants butylated hydroxyanisole or N,N-diphenyl-1,4-phenylenediamine or the iron chelators. Desferal or bathophenanthrolinedisulfonic acid. In contrast, both (S)-4-OH-OTA and lipid peroxidation showed a pH optimum of 7.0 and both activities were sensitive to inhibition by the above antioxidants and iron chelators. Lipid peroxidation was not involved in (S)-4-OH-OTA formation since addition of linoleic acid hydroperoxide to microsomes did not give rise to (S)-4-OH-OTA. Cytochrome P450 appeared to be essential since other hemoproteins like horseradish peroxidase and hemoglobin were ineffective in metabolizing OTA in the presence of hydroperoxides. The results suggest that (R)-4-OH-OTA is formed by normal mixed-function oxidation but that (S)-4-OH-OTA formation may involve free iron. It is likely that an active Fe2(+)-oxygen complex, formed via NADPH-cytochrome P450 reductase and cytochrome P450-dependent reduction of free Fe3+ followed by oxygen binding, serves as the species inducing lipid peroxidation and at least part of (S)-4-OH-OTA formation.

Alterations in ATP-dependent calcium uptake by rat renal cortex microsomes following ochratoxin A administration in vivo or addition in vitro.

A disruption of calcium homeostasis, leading to a sustained increase in cytosolic calcium levels, has been associated with cytotoxicity in response to a variety of agents in different cell types. We have observed that administration of a single high dose or multiple lower doses of the carcinogenic nephrotoxin ochratoxin A (OTA) to rats resulted in an increase of the renal cortex endoplasmic reticulum ATP-dependent calcium pump activity. The increase was very rapid, being evident within 10 min of OTA administration and remained elevated for at least 6 hr thereafter. The increase in calcium pump activity was inconsistent with previous observations that OTA enhances lipid peroxidation (ethane exhalation) in vivo, a condition known to inhibit the calcium pump. However, no evidence of enhanced lipid peroxidation was observed in the renal cortex since levels of malondialdehyde and a variety of antioxidant enzymes including catalase, DT-diaphorase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione S-transferase were either unaltered or reduced. In in vitro studies, addition of OTA to cortex microsomes during calcium uptake inhibited the uptake process although the effect was reversible. Preincubation of microsomes with NADPH had a profound inhibitory effect on calcium uptake but inclusion of OTA was able to reverse the inhibition. Changes in the rates of microsomal calcium uptake correlated with changes in the steady-state levels of the phosphorylated Mg2+/Ca(2+)-ATPase intermediate, suggesting that in vivo/in vitro conditions were affecting the rate of enzyme phosphorylation.

Mechanism of ochratoxin A stimulated lipid peroxidation.

Lipid peroxidation, measured as malondialdehyde formation or by oxygen uptake, was stimulated markedly by the mycotoxin ochratoxin A (OTA) in a reconstituted system consisting of phospholipid vesicles, the flavoprotein NADPH-cytochrome P450 reductase, Fe3+, EDTA and NADPH. Deletion of EDTA lowered the extent of lipid peroxidation but did not eliminate it. Fluorometric and spectrophotometric studies demonstrated the formation of a 1:1 Fe3(+)-OTA complex. The rate of reduction of Fe3+ to Fe2+ was enhanced markedly in the presence of OTA, and there was a further increase in the rate when EDTA was also included. The data indicate that OTA stimulates lipid peroxidation by complexing Fe3+ and facilitating its reduction. Subsequent to oxygen binding, an iron-oxygen complex of undetermined nature initiates lipid peroxidation. Free hydroxyl radicals appear not to participate in lipid peroxidation stimulated by Fe3(+)-OTA.

Lipid peroxidation as a possible cause of ochratoxin A toxicity.

Addition of the mycotoxin ochratoxin A (OA), a nephrotoxic carcinogen, to rat liver microsomes greatly enhanced the rate of NADPH or ascorbate-dependent lipid peroxidation as measured by malondialdehyde formation. NADPH-dependent lipid peroxidation in kidney microsomes was similarly enhanced by OA. The process required the presence of trace amounts of iron but cytochrome P-450 and free active oxygen species appeared not to be involved. The efficiency of several ochratoxins (ochratoxins A, B, C, alpha and O-methyl-ochratoxin C) to enhance lipid peroxidation was related to the presence and reactivity of the phenolic hydroxyl group. Furthermore, the ability of these ochratoxins to enhance lipid peroxidation in microsomes correlated precisely with their known toxicities in chicks. Administration of ochratoxin A to rats also resulted in enhanced lipid peroxidation in vivo as evidenced by a seven-fold increase in the rate of ethane exhalation. These results suggest that lipid peroxidation may play a role in the observed toxicity of ochratoxin A in animals; a mechanism is proposed. (Formula: see text). Ochratoxin A: X = Cl; R1 = R2 = R3 = R4 = H Ochratoxin B: X = H; R1 = R2 = R3 = R4 = H Ochratoxin C: X = Cl; R1 = R2 = R3 = H; = R4 = CH3 O-Methyl-ochratoxin C: X = Cl; R2 = R3 = H; R1 = R4 = CH3 (4R)-4-hydroxyochratoxin A: X = Cl; R1 = R3 = R4 = H; R2 = OH (4S)-4-hydroxyochratoxin A: X = Cl; R1 = R2 = R4 = H; R3 = OH Fig. 1. Chemical structures of the various ochratoxins.

Perturbation of liver microsomal calcium homeostasis by ochratoxin A.

The effect of ochratoxin A on hepatic microsomal calcium sequestration was studied both in vivo and in vitro. The rate of ATP-dependent calcium uptake was inhibited by 42-45% in ochratoxin A intoxicated rats as compared to controls. In the presence of NADPH, addition of ochratoxin A (2.5 to 100 microM) caused a concentration-dependent inhibition of calcium uptake (28-94%) by untreated rat liver microsomes. The rate of NADPH-dependent lipid peroxidation, measured as malondialdehyde formed, was also greatly enhanced by ochratoxin A. Various agents that inhibited ochratoxin A enhanced lipid peroxidation were also able to block the destruction of calcium uptake activity. Lipid peroxidation enhanced by ochratoxin A was also accompanied by leakage of calcium from calcium-loaded microsomes. These results suggest that ochratoxin A disrupts microsomal calcium homeostasis by an impairment of the endoplasmic reticulum membrane probably via enhanced lipid peroxidation.

Effect of cytochrome P450 induction on the metabolism and toxicity of ochratoxin A.

Liver microsomes from rats treated with various P450 inducers were examined for their ability to metabolize the mycotoxin ochratoxin A (OTA) to 4(R)-4-hydroxyochratoxin A (4R), the major metabolite, and 4(S)-4-hydroxyochratoxin A (4S), the minor metabolite. Pretreatment of rats with phenobarbital (PB), dexamethasone (DXM), 3-methylcolcanthrene (3MC) and isosafrole (ISF) greatly induced 4R formation. PB, DXM, 3MC, clofibrate (CLF) and ISF treatments also induced 4S formation. Isoniazid (INH) pretreatment primarily induced 4S formation. The pH optimum for 4R formation was found to be 6.0 with 3MC microsomes, and 6.5 with PB and DXM microsomes. For 4S formation, the pH optimum was 7.0. At the optimum pH (compared with pH 7.4), 4R formation increased 40-50% with PB and DXM microsomes but 8.0-fold with 3MC microsomes. Studies using the inhibitors metyrapone and alpha-naphthoflavone as well as monoclonal antibodies against various P450s suggested that at least the P450 isoforms IA1/IA2, IIB1 and IIIA1/IIIA2 are involved in 4R formation. Using urinary excretion of the enzymes alkaline phosphatase and gamma-glutamyl transferase as an index of renal damage, we observed that pretreatment of rats with PB, which induced hepatic P450 (P450II2B1), protected against OTA nephrotoxicity, whereas cobalt-protoporphyrin IX pretreatment, which decreased P450 levels, exacerbated OTA nephrotoxicity. Our results suggest that at least P450IIB1-dependent metabolism of OTA leads to its detoxication and that OTA itself may be toxic in some circumstances or that other pathways are responsible for its activation.

Comparison of the inhibitory effects of some compounds present in crude oils on rat platelet aggregation: role of intra- and extra- cellular calcium.

In vitro addition of some representative aliphatic, aromatic or heterocyclic compounds present in petroleum crude oils to washed rat platelets resulted in a concentration-dependent inhibition of aggregation induced by ADP or thrombin. Increasing concentration of extracellular Ca2+ did not alter the pattern of inhibition. ADP-induced intracellular Ca2+ mobilization was unaffected by most of the compounds tested. However, Ca2+ uptake was significantly inhibited when platelets were preincubated with these agents. This suggests that some components of crude oil may inhibit platelet aggregation by bringing about alterations in the platelet plasma membrane.

The hepatotoxic potential of a Prudhoe Bay crude oil: effect on mouse liver weight and composition.

The hepatotoxic properties of a Prudhoe Bay Crude Oil (PBCO) were evaluated in mice. Administration of PBCO (5.0 ml/kg body wt, daily for 2 days) to mice resulted in an increase in (i) liver wet and dry weight, (ii) hepatic total proteins, RNA, glycogen and total lipids, and (iii) individual lipids such as cholesterol, triglycerides and phospholipids. Hepatic protein biosynthesis, determined in vivo by administration of L-[14C]leucine was increased in PBCO exposed mice. The rate of 3H incorporation from 3H2O was significantly enhanced in liver fatty acids, cholesterol, triglycerides and thus ultimately in total lipids. Also, an increase in 3H incorporation was noticed in hepatic glycogen after PBCO administration. The results suggest that PBCO may induce hepatotoxicity by altering the intermediary metabolism of biochemical constituents.

Mechanisms of petroleum hydrocarbon toxicity: studies on the response of rat liver mitochondria to Prudhoe Bay crude oil and its aliphatic, aromatic and heterocyclic fractions.

The effect of Prudhoe Bay crude oil (PBCO) and its different fractions [aliphatic, aromatic, heterocyclic (NOS)] on the bioenergetic functions of isolated rat liver mitochondria were studied. A DMSO extract of PBCO inhibited state 3 respiration (in the presence of ADP) with either succinate or beta-hydroxybutyrate as substrate. The ascorbate-TMPD dependent state 3 respiration was not affected. Succinate dehydrogenase and beta-hydroxybutyrate dehydrogenase activities were also lost in the presence of the PBCO extract suggesting that inhibition of state 3 respiration may be due to blockage of the electron transport chain. Stimulation of state 4 respiration (in the absence of ADP) and of the oligomycin sensitive ATPase activity by the PBCO extract was observed. Fractionation of PBCO indicated that the aromatic fraction was mainly responsible for its inhibitory effects. By comparison, the heterocyclic fraction had weak inhibitory properties while the aliphatic fraction was essentially inactive. It is concluded that the aromatic components of PBCO inhibit mitochondrial respiration and oxidative phosphorylation mainly through impairment of the mitochondrial membrane and inhibition of beta-hydroxybutyrate and succinate dehydrogenase supported electron transfer activities of the respiratory chain.

Dietary administration of 2(3)-t-butyl-4-hydroxyanisole elevates mouse liver microsome-mediated DNA binding and mutagenicity of aflatoxin B1.

Administration of the phenolic antioxidant 2(3)-t-butyl-4-hydroxyanisole (BHA) to mice resulted in a 2-3-fold increase in the liver microsome catalyzed irreversible binding of aflatoxin B1 (AFB1) to calf thymus DNA and up to a 5-fold increase in the ability to induce mutations in Salmonella typhimurium TA98. Maximum induction of AFB1 binding to DNA occurred after 2 days of BHA administration whereas cytosolic glutathione S-transferase was maximally induced (6-fold) only after 10 days of BHA feeding. The induction of a new cytochrome P-450 species was indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and an enhanced sensitivity to inhibition by metyrapone and alpha-naphthoflavone. Addition of control cytosol (containing glutathione S-transferase) + glutathione to control microsomes decreased AFB1 binding to DNA by 26%. However, replacement of control cytosol by BHA cytosol which contained 6 times more glutathione S-transferase only marginally enhanced the inhibition to 38%. These data suggest that BHA may exert its effect in the liver primarily through an alteration of the cytochrome P-450 dependent activation process although an increase in the conjugation of reactive metabolite may play a contributory role.

Peroxidase-catalyzed benzidine binding to DNA and other macromolecules.

[14C]Benzidine is rapidly oxidized by a peroxidase/H2O2 system to products which bind irreversibly to DNA. The presence of exogenous DNA also prevented benzidine polymerization to 'benzidine brown' and azobenzidine. Two molar equivalents of H2O2 were required to oxidize the benzidine and achieve maximal DNA binding. Furthermore, 95% of the benzidine was trapped and 36 nmol benzidine was bound per mg DNA. Polyriboguanylic acid was as effective as DNA in binding benzidine, but polyriboadenylic acid, polyribouridylic acid and polyribocytidylic acid were much less effective. Binding of [14C]benzidine correlated well with the absorbance at 295 nm and 390 nm of the modified DNA or various synthetic homopolymers of ribonucleotides isolated from the reaction mixture. The peroxidase/H2O2 system also catalyzed the binding of dichlorobenzidine, o-tolidine and o-dianisidine to DNA but 3,5,3',5'-tetramethylbenzidine, a non-carcinogen, did not bind. The binding could be prevented by various biological hydrogen donors, thiols, or phenolic antioxidants. The mechanisms for DNA protection were investigated; the oxidized benzidine species involved in binding can be reduced with ascorbate, NADPH, or thiols, and trapped by thiols or phenolic antioxidants to form conjugates or adducts.

Mechanisms of petroleum hydrocarbon toxicity: functional changes in rat liver mitochondria after exposure to a Prudhoe Bay Crude Oil.

Administration of a Prudhoe Bay Crude Oil (PBCO) to rats (5 ml/kg/day for 2 days) resulted in a 30-35% inhibition of liver mitochondrial state 3 respiration with beta-hydroxybutyrate or succinate as substrates. beta-Hydroxybutyrate dehydrogenase, succinate dehydrogenase, NADH oxidase and succinate oxidase activities were also decreased in PBCO-treated rats. A significant increase in latent ATPase activity was also observed. Our results suggest that PBCO exerts its effects on liver mitochondria through the inhibition of beta-hydroxybutyrate- and succinate-supported electron transfer activities and by impairment of the mitochondrial membrane.

Hydrocarbon-based oils as inducers of cutaneous aryl hydrocarbon hydroxylase.

Petroleum-based oils applied to the skin of rats induced cutaneous aryl hydrocarbon hydroxylase activity. A heavy oil like Kuwait Crude induced benzo(a)pyrene (BaP) hydroxylase and diphenyloxazole (PPO) hydroxylase activities to levels obtained with Aroclor 1254, while a light oil like Fuel Oil No. 2 was less effective. Intraperitoneal injection of these oils to rats also induced hepatic aryl hydrocarbon hydroxylase activities. Inhibition of these activities by alpha-naphthoflavone, but not by metyrapone, suggests that the cytochrome induced is of the P448 type.

Embryotoxic evaluation of a Prudhoe Bay crude oil in rats.

The embryotoxic potential of a Prudhoe Bay crude oil (PBCO) was investigated in rats. PBCO was administered orally to pregnant rats as (i) a single dose on various gestation days, (ii) a single variable dose on gestation day 6, or (iii) as daily doses from day 6 to day 17 of pregnancy. PBCO administered during the earlier stages of pregnancy (day 3, 6 or 11) but not during the later stages, affected the reproductive performance of pregnant rats by significantly increasing the number of resorptions including fetal death and by decreasing the fetal weight. A dose-dependent increase in fetomortality was also observed. Multiple exposure to low levels of crude oil also caused a significant reduction in maternal body weight besides other embryotoxic changes.

Metabolism of 2,6-dimethylnaphthalene by rat liver microsomes and effect of its administration on glutathione depletion in vivo.

Metabolism of the environmental contaminant 2,6-dimethylnaphthalene (2,6-DMN) by rat liver microsomes and an NADPH-regenerating system led to the formation of three ring oxidation metabolites--2,6-dimethyl-3-naphthol, 2,6-dimethyl-3,4-naphthoquinone, and 3,4-dihydro-3,4-dihydroxy-2,6-dimethylnaphthalene--and one side chain oxidation metabolite--2-hydroxymethyl-6-methylnaphthalene. In addition, one metabolite remained unidentified. Pretreatment of rats with phenobarbital, 3-methylcholanthrene, Prudhoe Bay crude oil, or 2,6-DMN enhanced the rate of microsomal metabolism of 2,6-DMN 2-6-fold and significantly altered the metabolite profile. Liver microsomes from variously pretreated rats also enhanced the irreversible binding of 2,6-DMN[8-14C]N to microsomal protein. This binding to protein was inhibited by glutathione in a concentration-dependent manner. In vivo administration of 2,6-DMN to untreated rats led to a time-dependent depletion of hepatic glutathione levels. Both the rate and the extent of glutathione depletion were significantly enhanced in 3-methylcholanthrene-pretreated rats but not in phenobarbital-pretreated rats.

The role of heme metabolism during the induction of hepatic and renal cytochrome P-450 levels and drug-metabolizing enzymes in rats by a Prudhoe Bay crude oil.

Administration of Prudhoe Bay crude oil (PBCO) to rats resulted in a dose-related increase in liver weight; rapid and marked increase in the activity of hepatic delta-aminolevulinate synthetase, the initial and rate-limiting enzyme in the heme biosynthetic pathway; rapid decline in the activity of hepatic heme oxygenase, the rate-limiting enzyme of heme catabolism; and more gradual increase in the levels of hepatic cytochrome P-450 and some mixed-function oxidase activities such as benzo[a]pyrene hydroxylase and 7-ethoxyresorufin-O-deethylase. PBCO treatment also increased renal cytochrome P-450 levels and mixed-function oxidase activities; however, delta-aminolevulinate synthetase and heme oxygenase activities were unchanged. This suggests that different regulatory mechanism(s) may be involved in renal heme metabolism and induction of monoxygenase system.