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Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.

The use of the extremely effective anthracycline antitumor drugs adriamycin and daunomycin is limited by a severe, dose-dependent cardiomyopathy. Anthracycline-induced toxicity has been proposed to involve iron-dependent oxidative damage to biological macromolecules yet little is known regarding the availability of physiologic iron. We now report that, in the presence of NADPH-cytochrome P-450 reductase, these drugs undergo redox cycling to generate superoxide which mediates a slow, reductive release of iron from ferritin, the major intracellular iron storage protein. Anaerobically, the semiquinone free radical forms of adriamycin and daunomycin catalyze a very rapid, extensive release of iron from ferritin. In contrast, diaziquone, an aziridinyl quinone antitumorigenic agent which is less cardiotoxic, is unable to release iron from ferritin. Thus, the present studies suggest that the cardiomyopathy observed with the anthracyclines, and perhaps their antineoplastic activity as well, may be related to their ability to delocalize tissue iron, thereby contributing to the formation of strong oxidants capable of damaging critical cellular constituents.

A lipid peroxidation system consisting of phospholipid liposomes, paraquat, ADP, and NADPH-cytochrome P450 reductase was constituted using ferritin as the sole source of iron. Lipid peroxidation was completely inhibited by superoxide dismutase, essentially not affected by mannitol, but markedly stimulated by catalase. Similar effects of these scavenging agents were observed in incubations void of ADP. These data suggest that O2-, produced by the redox cycling of paraquat, can release iron from ferritin and thereby promote lipid peroxidation. The effects of catalase and mannitol suggest that the initiation of peroxidation, in either the presence or absence of ADP, is not significantly dependent upon the hydroxyl radical produced via an iron-catalyzed Haber-Weiss reaction.

The white rot fungus Phanerochaete chrysosporium degraded DDT [1,1,-bis(4-chlorophenyl)-2,2,2-trichloroethane], 3,4,3',4'-tetrachlorobiphenyl, 2,4,5,2',-4',5'-hexachlorobiphenyl, 2,3,7,8-tetrachlorodibenzo-p-dioxin, lindane (1,2,3,4,5,6-hexachlorocylohexane), and benzo[a]pyrene to carbon dioxide. Model studies, based on the use of DDT, suggest that the ability of Phanerochaete chrysosporium to metabolize these compounds is dependent on the extracellular lignin-degrading enzyme system of this fungus.

The potential for iron bound to transferrin to be released and promote the peroxidation of phospholipid liposomes was investigated using ADP as a low molecular weight chelator and superoxide generated by the xanthine/xanthine oxidase system as the reducing agent. Lipid peroxidation in this system was dependent upon transferrin as the source of iron; increasing the transferrin concentration resulted in increased rates of lipid peroxidation. Increasing the xanthine oxidase activity also caused increased rates of peroxidation. Catalase stimulated rates of peroxidation at all xanthine oxidase activities tested. Conditions resulting in the most rapid release of iron from transferrin (low pH, high ADP) did not promote the greatest rates of lipid peroxidation, indicating that at neutral pH, rates of lipid peroxidation may be limited by the availability of iron. It is concluded that transferrin is not a likely source of iron for catalysis of deleterious biological oxidations such as lipid peroxidation in vivo.

Ferritin was found to promote the peroxidation of phospholipid liposomes, as evidenced by malondialdehyde formation, when incubated with xanthine oxidase, xanthine, and ADP. Activity was inhibited by superoxide dismutase but markedly stimulated by the addition of catalase. Xanthine oxidase-dependent iron release from ferritin, measured spectrophotometrically using the ferrous iron chelator 2,2'-dipyridyl, was also inhibited by superoxide dismutase, suggesting that superoxide can mediate the reductive release of iron from ferritin. Potassium superoxide in crown ether also promoted superoxide dismutase-inhibitable release of iron from ferritin. Catalase had little effect on the rate of iron release from ferritin; thus hydrogen peroxide appears to inhibit lipid peroxidation by preventing the formation of an initiating species rather than by inhibiting iron release from ferritin. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide was used to observe free radical production in this system. Addition of ferritin to the xanthine oxidase system resulted in loss of the superoxide spin trap adduct suggesting an interaction between superoxide and ferritin. The resultant spectrum was that of a hydroxyl radical spin trap adduct which was abolished by the addition of catalase. These data suggest that ferritin may function in vivo as a source of iron for promotion of superoxide-dependent lipid peroxidation. Stimulation of lipid peroxidation but inhibition of hydroxyl radical formation by catalase suggests that, in this system, initiation is not via an iron-catalyzed Haber-Weiss reaction.

The interaction of isosafrole, 3,4,5,3',4',5'-hexabromobiphenyl (HBB) and hexachlorobiphenyl (HCB) with cytochrome P-450d was evaluated by characterization of estradiol 2-hydroxylase activity. Displacement of the isosafrole metabolite from microsomal cytochrome P-450d derived from isosafrole-treated rats resulted in a 160% increase in estradiol 2-hydroxylase. The increase was fully reversed by incubation with 1 microM HBB. Although isosafrole is capable of forming a complex with many different cytochrome P-450 isozymes, it appears to bind largely to cytochrome P-450d in vivo as was demonstrated by measuring the enzymatic activity of microsomal cytochromes P-450b, P-450c, and P-450d from isosafrole-treated rats. When estradiol 2-hydroxylase was measured in rats treated with increasing doses of HCB, there was a gradual decrease in microsomal enzyme activity despite a 20-fold increase in cytochrome P-450d. The ability of cytochrome P-450d ligands to stabilize the enzyme was investigated in two ways. First, cytochromes P-450c and P-450d were quantitated immunochemically in microsomes from rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a dose which maximally induced total cytochrome P-450, followed by a single dose of a second inducer. The specific content of cytochrome P-450d was significantly increased when isosafrole or HCB was the second inducer but not when 3-methylcholanthrene was the second inducer. Second, the relative turnover of cytochrome P-450d was measured by the dual label technique. Following TCDD treatment, microsomal protein was labeled in vivo with [3H]leucine, the second inducer was given and protein was again labeled 3 days later with [14C]leucine. A higher ratio of 3H/14C in the cytochrome P-450d from isosafrole + TCDD- and HCB + TCDD-treated rats relative to TCDD (control)-treated rats suggested that isosafrole and HCB were able to retard the degradation of cytochrome P-450d, presumably by virtue of being tightly bound to the enzyme.

Pathologic effects of 2,2',4,4',5,5'-hexabromobiphenyl (HBB), 2,3',4,4',5,5'-HBB, and a commercial mixture of polybrominated biphenyls (PBB) were compared in White leghorn cockerels. Diets containing 1, 10, or 100 ppm PBB, 4 or 10 ppm 2,3',4'4',5,5'-HBB, or 10 or 62 ppm 2,2',4,4',5,5'-HBB were fed for 28 days. Doses of 10 ppm of each chemical were used to provide a direct comparison of toxicity. Since nearly 4% of PBB consists of 2,3',4,4',5,5'-HBB and approximately 62% consists of 2,2',4,4',5,5'-HBB, effects of doses of 4 and 62 ppm, respectively, were compared with effects of 100 ppm of PBB to determine if either of the congeners were mainly responsible for the pathologic effects caused by the mixture. Liver weights were increased in cockerels fed diets containing 62 ppm of 2,2',4,4',5,5'-HBB or 10 or 100 ppm PBB. Hepatocytes were enlarged and vacuolated and lymphoid cells of the bursa of Fabricius were depleted by 10 ppm 2,3',4,4',5,5'-HBB, ppm 2,2',4,4',5,5'-HBB, and 10 or 100 ppm PBB. These dietary concentrations caused ultrastructural changes in hepatocytes consisting of vacuolation, increased smooth endoplasmic reticulum, swollen mitochondria, and disruption of mitochondrial cristae. When either of the congeners were given in concentrations relative to their concentrations in PBB, they were less toxic than the mixture. When concentrations in diets were equal, PBB caused more severe effects than 2,3',4,4',5,5'-HBB. The least effects were seen with 2,2',4,4',5,5'-HBB. Results indicate that the two congeners chosen for study are not individually as toxic as the parent mixture.

The irradiation of 2,4,5,2',4',5'-hexabromobiphenyl (2,4,5-HBB) by ultraviolet light created a mixture of lower brominated polybrominated biphenyl (PBB) congeners. Three photoproducts, 2,4,5,3',4'-pentabromobiphenyl (-PBB), 2,4,5,2',5'-PBB, and 3,4,3',4'-tetrabromobiphenyl (3,4-TBB), as well as 2,4,5-HBB and the photolyzed 2,4,5-HBB mixture, were administered to rats as a single ip injection (90 mg/kg, except 3,4-TBB, which was given at 2 mg/kg) 2 weeks before sacrifice. All treatments except 3,4-TBB induced NADPH-cytochrome P-450 reductase and aminopyrine-N-demethylase activities while all treatments except 2,4,5-HBB induced ethoxyresorufin-O-deethylase and UDP-glucuronosyltransferase activities. Thymus to body weight and spleen to body weight ratios were unchanged compared to controls for all treatments whereas an increase in the liver weights was observed for all treatment groups. Histologic examination revealed that the photolyzed 2,4,5-HBB mixture caused moderate to severe hepatocyte enlargement. Results of tissue analysis for the pure PBB congeners indicated that 2,4,5,2',5'-PBB and 3,4-TBB were metabolized in vivo and this was confirmed by in vitro metabolism studies. The results revealed that the photolyzed 2,4,5-HBB mixture caused a mixed-type induction of hepatic drug-metabolizing enzymes. This is most likely due to the effect of 2,4,5-HBB and toxic congeners formed during the irradiation of 2,4,5-HBB. 2,4,5,3',4'-PBB, which is toxic and apparently not metabolized, is believed to be the major congener contributing to the increased toxicity of the photolyzed 2,4,5-HBB mixture since 3,4-TBB was metabolized and appeared not to be as potent as inducer of aryl hydrocarbon hydroxylase activity.

Some toxicological and pharmacological effects of 2,4,5,2',5'-penta- (congener 1), 2,3,4,2',4',5'-hexa- (congener 5), 2,4,5,3',4',5'-hexa- (congener 6), 2,3,4,5,3',4',-hexa- (congener 7), and 2,3,4,5,2',3',4'-heptabromobiphenyl (congener 9) were evaluated in male rats given a single 90 mg/kg ip injection and killed seven days later. Only congener 7 depressed body weight gain, spleen and thymus weights, and caused severe histopathological changes in the thymus. Congener 7 caused the largest increase in liver weight and the most changes in liver pathology while congener 1 failed to enlarge this organ and caused the mildest ultrastructural changes. Liver microsomes were isolated and evaluated for enzyme induction from all treated rats except those administered congener 6, which was previously identified as a mixed-type enzyme inducer (Dannan et al., 1978b). All congeners increased the liver microsomal cytochrome P-450 content, but only congener 7 shifted the carbon monoxide difference spectrum absorption maximum to 448.0 nm. The microsomal ethyl isocyanide difference spectrum 455/430 nm ratio was increased the most by congener 7 (3 fold). All congeners increased cytochrome P-450 reductase and microsomal epoxide hydrase activities by nearly 1.5-3 fold. Congener 7 failed to induce aminopyrine-N-demethylase activity but the remaining congeners increased it by 2 fold. Congener 7 was the most effective inducer of benzo[a]pyrene hydroxylase and p-nitrophenol UDP-glucuronyl transferase. These results add to the suggestion that the presence of an ortho halogen on a polyhalogenated biphenyl does not completely abolish toxicity or 3-methylcholanthrene-type microsomal enzyme induction.

The accidental contamination of dairy cattle feed in Michigan in 1973-74 with polybrominated biphenyls (PBB) led to the contamination of cattle and people consuming their products. This led to an extensive animal and product monitoring and disposal program conducted by the Michigan Department of Agriculture and the Department of Natural Resources. It also led to several studies of the people of Michigan, extensive research on the chemicals, and an unprecedented establishment by the Legislature of a Toxic Substance Control Commission. Only a few relatively minor components of the PBB mixture that contaminated Michigan are metabolized and another group of minor components seem responsible for the toxicity, which, similar to that caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), includes induction of microsomal enzymes, liver hypertrophy, thymic involution, porphyria, anorexia and chloracne. PBB were found to produce the "dioxin-like" toxicity with roughly 0.01% the potency of TCDD. Both non-toxic as well as toxic congeners were found to be tumor promotors. To date it is impossible to unequivocally conclude that any human health effects can be attributed to PBB. The Toxic Substance Control Commission was established as an independent oversight body with responsibility to gather information, investigate, coordinate and make recommendations concerning toxic substances and the handling of toxic substances incidents. The Commission has declared two toxic substances emergencies and made several recommendations for regulating and solving toxic substances problems but its major activities have evolved towards a role as an environmental ombudsman.

The effects of nonadecafluoro-n-decanoic acid (NDFDA) on serum retinol levels and hepatic retinyl palmitate hydrolase (RPH) activity were investigated in male Sprague-Dawley rats given a single intraperitoneal (IP) dose of 0, 50, or 100 mg/kg NDFDA and sacrificed at two, eight, or 11 days. Treated animals exhibited depressed serum retinol levels, lymphoid involution, and failure to gain weight in proportion to the dose. Hepatic RPH activities were depressed in both treatment groups at all time points and correlated with serum retinol levels. Hepatic retinol levels were also depressed by Day 11. Extraction of hepatic homogenates with acetone removed NDFDA and increased RPH activities twofold and threefold for the low- and high-dose groups, respectively. Analysis of partially purified RPH showed both NDFDA and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to be noncompetitive inhibitors: KI = 450 and 750 microM, respectively. We conclude that NDFDA causes a decrease in the mobilization of vitamin A from the liver by noncompetitive inhibition of RPH.

Cell damage initiated during ischemia matures during reperfusion. Mechanisms involved during reperfusion include the effects of arachidonic acid and its oxidative products prostaglandins and leukotrienes, reperfusion tissue calcium overloading, and damage to membranes by lipid peroxidation. Lipid peroxidation occurs by oxygen radical mechanisms that require a metal with more than one ionic state (transitional metal) for catalysis. We have shown that cellular iron is delocalized from the large molecules where it is normally stored to smaller chemical species during postischemic reperfusion. Postischemic lipid peroxidation is inhibited by the iron chelator deferoxamine. Intervention in the reperfusion injury of membranes by chelation of transitional metals is a new and promising therapeutic possibility for protection of the heart and brain.

Since its introduction in 1960, CPR has evolved into a complex program involving not only the medical community but also the lay public. Currently, program activities include instruction of the lay public in basic life support techniques, development and deployment of emergency medical systems, recommendations for drug protocols for advanced cardiac life support and, most recently, introduction of new methods for tissue protection following resuscitation. After 25 years of experience, we are beginning to understand the pathophysiology of tissue ischemia during cardiac arrest and the interventions required to improve chances of survival and quality of life of the cardiac arrest victim. Recent data in the literature suggest that modification of certain interventions in the resuscitation program may be needed. The poor neurologic outcomes with prolonged standard CPR show that it is not protective after 4 to 6 minutes of cardiac arrest. Modifications to this technique, including SVC-CPR or IAC-CPR, have not been shown to increase resuscitability or hospital discharge rates. Human studies of open-chest cardiac massage are needed to evaluate this option. Defibrillation is the definitive treatment for ventricular fibrillation. Greater emphasis should be placed on the earliest possible delivery of this treatment modality. Computerized defibrillators may provide greater and earlier access to defibrillation in the homes of patients at high risk of ventricular fibrillation. They may also be applicable by untrained public service personnel (police and firemen), individuals in geographically inaccessible areas (aircraft), or emergency medical technicians in rural areas where skill retention is a significant problem. Calcium has no proved benefit in cardiac resuscitation. There is biochemical evidence that it may be harmful in brain resuscitation. Its use in resuscitation should be discontinued. The dose of epinephrine currently advocated in the ACLS protocols may be inadequate to increase aortic diastolic pressure and coronary and cerebral perfusion pressures and thus aid resuscitation. Animal studies indicate that substantial increases in the current dosage are needed to achieve these effects. Human studies are needed to verify these results. A role for calcium antagonists in the treatment of postarrest encephalopathy has been demonstrated in animals and is currently undergoing clinical trials. Iron-dependent lipid peroxidative cell membrane injury may be important in the pathogenesis of postarrest encephalopathy. Animal studies suggest that the iron chelator deferoxamine may have a significant therapeutic role in the treatment of postarrest encephalopathy.

A study was conducted to determine aflatoxin levels in the tissues of broiler chickens that had been fed a diet containing 2057 micrograms aflatoxin B1 and 1323 micrograms aflatoxin B2/kg for 35 days. Results showed that aflatoxins were deposited in all tissues. The highest levels of aflatoxins were present in the gizzards, livers and kidneys. There was evidence that the high levels of aflatoxins B1 and B2 in the gizzards might have been caused by contamination by the gizzard contents during the slaughtering process. After feeding the aflatoxin-contaminated diet for 35 days, mean values for the combined aflatoxins were less than 3 micrograms/kg of tissue. Four days after withdrawal of the aflatoxin-contaminated ration, there were no detectable amounts of aflatoxins in any of the tissues. The results indicate that broiler chickens rapidly clear aflatoxins from their tissues once they are transferred to an aflatoxin-free diet.

When certain ferrous chelates are added to lipid, peroxidation of the lipid occurs following a short lag. This suggests that a product of ferrous autoxidation is required to initiate lipid peroxidation. This autoxidation product is apparently ferric iron, rather than the oxygen radicals which also result from ferrous autoxidation. Studies with oxy-radical scavengers and catalase suggest that O2-., H2O2, or the .OH are not involved in the initiation reactions, therefore, we propose that a ferrous-dioxygen-ferric chelate complex may be the initiating species.

The possible involvement of hydrogen peroxide (H2O2)-derived hydroxyl radical (.OH) in lignin degradation ([14C]lignin leads to 14CO2) by Phanerochaete chrysosporium was investigated. When P. chrysosporium was grown in low nitrogen medium (2.4 mM N), an increase in the specific activity for H2O2 production in cell extracts was observed to coincide with the appearance of ligninolytic activity and both activities appeared after the culture entered stationary phase. The production of .OH in ligninolytic cultures of P. chrysosporium was demonstrated by alpha-keto-gamma-methiolbutyric acid-dependent formation of ethylene. Hydrogen peroxide-dependent .OH formation was also shown in cell extracts of ligninolytic cultures. The radical species was demonstrated to be .OH by the .OH-dependent hydroxylation of p-hydroxybenzoic acid to form protocatechuic acid and by using 5,5-dimethyl-1-pyrroline-N-oxide and detecting the production of the nitroxide radical of 5,5-dimethyl-1-pyrroline-N-oxide by EPR. These reactions were inhibited by .OH-scavenging agents and were stimulated when azide was added to inhibit endogenous catalase. Lignin degradation by P. chrysosporium was markedly suppressed in the presence of the .OH-scavenging agents mannitol, benzoate, and the nonspecific radical scavenging agent butylated hydroxytoluene. The above results indicate that .OH derived from H2O2 is involved in lignin biodegradation by P. chrysosporium.

NADPH-dependent lipid peroxidation occurs in two distinct sequential radical steps. The first step, initiation, is the ADP-perferryl ion-catalyzed formation of low levels of lipid hydroperoxides. The second step, propagation, is the iron-catalyzed breakdown of lipid hydroperoxides formed during initiation generating reactive intermediates and products characteristic of lipid peroxidation. Propagation results in the rapid formation of thiobarbituric acid-reactive material and lipid hydroperoxides. Propagation can be catalyzed by ethylenediamine tetraacetate-chelated ferrous ion, diethylenetriamine pentaacetic acid-chelated ferrous ion, or by ferric cytochrome P-450. However, cytochrome P-450 is destroyed during propagation.

Polybrominated biphenyls (PBBs) cause a mixed-type (phenobarbital- plus 3-methylcholanthrene-like) induction of liver microsomal drug metabolizing enzymes in rats. However, 2,2',4,4',5,5'-hexabromobiphenyl and 2,2',3,4,4',5,5'-heptabromobiphenyl, which together comprise less than 80% of PBBs (FireMaster), were shown to be strictly phenobarbital-type inducers. Other components (unidentified) must therefore cause the 3-methylcholanthrene-like effects. The potential for PBBs to exert effects on neonates through milk was examined. Lactating rats were fed 0, 0.1, 1.0, or 10 ppm FireMaster for the 18 days following delivery, at which time mothers and most pups were sacrificed. Pups nursing from mothers fed 10 ppm PBBs showed significant increases in liver weights and microsomal protein, and both mothers and pups had increased cytochrome P-450, aminopyrine demethylation, benzo[a]pyrene hydroxylation, and UDP-glucuronyltransferase. Pups nursing from rats fed 1.0 ppm had increases in microsomal protein, cytochrome P-450, aminopyrine demethylation, and benzo[a]pyrene hydroxylation, while their mothers were unaffected. Several pups from the 0, 0.1, and 1.0 ppm groups were maintained on their mother's diets, raised, and allowed to mate. Their pups showed much the same responses to PBBs as did the original group of pups. The effects on both generations of adult female rats were also comparable. PBBs cause a mixed-type induction in both lactating rats and their nursing pups; PBB components responsible for both aspects of this induction must be transmitted through milk. Nursing rats are approximately tenfold more sensitive to the effects of PBBs in their mother's diets than are the dams. The approximate no-effect level for microsomal induction in nursing rats is 0.1 ppm PBBs in the diet of the adult.

A commercial preparation of polybrominated biphenyls (Firemaster BP-6) was fractionated by preferential acetone solubilization and a number of PBB congeners were purified from the resulting fractions by repeated recrystallization, alumina adsorption column chromatography, and reversed-phase Lipidex-500 column chromatography. The applicability of reversed-phase Lipidex-5000 chromatography for the separation of highly structurally similar brominated biphenyl congeners that otherwise could not be separated by alumina chromatography or recrystallization was demonstrated. In total, seven congeners were purified, including one whose structure was assigned as 2,3,4,5,3',4'-hexabromobiphenyl.

Lipid peroxidation is thought to be a major contributing factor in neurological injury following cardiac arrest. Because iron availability is a prerequisite for lipid peroxidation, this experiment was designed to examine the natural time course of iron release, lipid peroxidation, and cerebral polyunsaturated fatty acid content following a 15-minute cardiac arrest in dogs. Large mongrel dogs were anesthetized with ketamine and halothane and divided into three groups of five each. In two groups, cardiac arrest was induced with KCl. After 15 minutes of cardiac arrest, the dogs were resuscitated by five minutes of internal cardiac massage, epinephrine, bicarbonate, and internal defibrillation. All ten dogs were resuscitated and supported by a standard intensive care protocol until tissue harvest. A 3-g portion of parietal cerebral cortex was obtained from the nonischemic dogs (n = 5), or at two hours (n = 5), or eight hours (n = 5) after resuscitation. Total tissue iron was measured by an atomic emission spectrometer; low molecular weight species (LMWS) iron by the o-phenanthroline test on an ultrafiltered sample; and lipid peroxidation by both the thiobarbituric acid test (TBARS) and determination of the tissue content of lipid double bonds, calculated by first fractionating the lipids by gas-liquid chromatography and then measuring the double bonds in each fraction by spectrometry and summing the results. Univariate ANOVA demonstrated all variables except total tissue iron to have significance at P less than .02. At two hours of reperfusion, LMWS iron and TBARS were significantly elevated above nonischemic control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Partially-reduced forms of dioxygen or "oxy-radicals" (superoxide, O2-/HO2; hydrogen peroxide, H2O2; hydroxyl radical X OH) and oxidants of comparable reactivity are implicated in an increasing number of physiological, toxicological, and pathological states. Transition metal catalysis is recognized as being integral to the generation and the reactions of these activated oxygen species. Factors such as pH and chelation govern the reactivity of the transition metals with dioxygen and "oxy-radicals" and therefore influence the apparent mechanisms by which oxidative damage to phospholipids, DNA, and other biomolecules is initiated. In biological systems the concentrations of redox-active transition metals capable of catalyzing these reactions appears to be relatively low. However, under certain conditions metal storage and transport proteins (ferritin, transferrin, ceruloplasmin, etc.) may furnish additional redox active metals.

The initiation of lipid peroxidation by Fe2+ and H2O2 (Fenton's reagent) is often proposed to be mediated by the highly reactive hydroxyl radical. Using Fe2+, H2O2, and phospholipid liposomes as a model system, we have found that lipid peroxidation, as assessed by malondialdehyde formation, is not initiated by the hydroxyl radical, but rather requires Fe3+ and Fe2+. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide and the bleaching of para-nitrosodimethylaniline confirmed the generation of the hydroxyl radical in this system. Accordingly, catalase and the hydroxyl radical scavengers mannitol and benzoate efficiently inhibited the generation and the detection of hydroxyl radical. However, catalase, mannitol, and benzoate could either stimulate or inhibit lipid peroxidation. These unusual effects were found to be consistent with their ability to modulate the extent of Fe2+ oxidation by H2O2 and demonstrated that lipid peroxidation depends on the Fe3+:Fe2+ ratio, maximal initial rates occurring at 1:1. These studies suggest that the initiation of liposomal peroxidation by Fe2+ and H2O2 is mediated by an oxidant which requires both Fe3+ and Fe2+ and that the rate of the reaction is determined by the absolute Fe3+:Fe2+ ratio.

The metabolism of polybrominated biphenyls (PBBs) was studied in vitro by using rat liver microsomes in the presence of NADPH and atmospheric O2. Quantitative recoveries of all PBBs were obtained after incubations with control or 3-methylcholanthrene (MC) induced microsomes. Of the twelve major components, losses of only peaks 1 (2,4,5,2',5'-pentabromobiphenyl) and 3 (a hexabromobiphenyl) were observed following incubations with microsomes from phenobarbital (PB)- or PBBS- pretreated rats. Of seven structurally identified PBB components, only peak 1 has a bromine-free para position. Peaks 1, 2, and 5 all have two adjacent unsubstituted carbons, yet only peak 1 is metabolized. Of two dibromobiphenyl model compounds studied, the 2,2'-congener was very rapidly metabolized by PB-induced microsomes whereas its 4,4'-isomer was not. These results suggest that the presence of a free para position is required for the metabolism of brominated biphenyls. Of lesser importance appears to be the number of bromines or the availability of two adjacent unsubstituted carbons. In vivo evidence for the metabolism of peaks 1 and 3 was also provided by their drastically diminished levels in liver and milk extracts. When a 14C-PBB mixture consisting almost exclusively of peaks 4 (2,4,5,2',4',5'-hexabromobiphenyl) and 8 (2,3,4,5,2',4',5'-heptabromobiphenyl) was incubated with PB- or PBBs- induced microsomes and NADPH, only traces of radioactivity remained with the microsomes after extensive extraction. However, less radioactivity was bound to microsomes from MC pretreated or especially control rats. No radioactivity was bound to exogenous DNA included in similar microsomal incubations, regardless of the type of microsomes used. Under the same conditions, [3H]-benzo[a]pyrene metabolites were bound to DNA, and PBB-induced microsomes enhanced this binding more than six-fold.

The interaction between dietary concentrations of vitamin A and PBB was evaluated in two experiments. In the first experiment, male weanling rats were used in a 2 X 3 factorial experiment. Concentrations of PBB were 0, 10, or 100 mg/kg of diet and diets were either vitamin A-deficient or were supplemented with 3000 IU vitamin A palmitate/kg of diet. In the second experiment the design was similar except that two vitamin A-supplemented diets were used, one containing 3,000 IU/vitamin A palmitate/kg of diet and the other 30,000 IU. Diets contained either 0 or 100 mg of PBB/kg. Clinical signs of vitamin A deficiency and mortality occurred early in rats fed vitamin A-deficient diets containing 100 mg of PBB/kg. Vitamin A supplementation provided partial protection against decreased weight gain associated with PBB. Decreases in thymic weight associated with PBB toxicosis were prevented by supplementation with vitamin A. Massive enlargement of the common bile duct occurred in rats fed a vitamin A-deficient diet containing 100 mg of PBB/kg. Histologically, this lesion consisted of extensive hyperplasia. A significant decrease in retinol concentrations in the sera was recorded in rats fed vitamin A-deficient diets containing 100 mg of PBB/kg. Interaction between vitamin A deficiency and PBB toxicosis affected vitamin A metabolism as manifested by the appearance of appreciable amounts of retinyl acetate in the liver vitamin A profile. These results suggest an interaction between PBB toxicity and vitamin A and emphasize the importance of nutritional factors such as vitamin A in assessment of PBB toxicosis.