Enzyme immunoassay for a urinary metabolite of 4-hydroxynonenal as a marker of lipid peroxidation.

Free radical reactions are involved in the pathogenesis of numerous diseases, so there is a real need to develop biomarkers that reflect these reactions in vivo. 4-Hydroxy-2-nonenal (HNE) is a major product of the lipid peroxidation process that is a consequence of free radical reactions. We present here the development and validation of an enzyme immunoassay (EIA) of the major urinary metabolite of HNE, namely 1,4-dihydroxynonane-mercapturic acid (DHN-MA). EIA allowed direct measurement of DHN-MA in rat urine with good sensitivity (0.02 ng/ml) and precision (intraassay CV = 5.7%). Recovery was complete (99-102%). Cross-reactivity was very low with 1,4-dihydroxynonene and with different mercapturic acids except with one other HNE urinary metabolite. Good correlation (EIA = 0.79 x LC/MS + 14.03, r = 0.877, p < 10(-8)) was obtained between EIA and liquid chromatography/mass spectrometry (LC/MS) quantitation when analyzing urine samples of rats with different oxidative status, due to treatment with either BrCCl(3) or trinitrobenzene sulfonic acid, which are known to induce hepatic lipid peroxidation or colon inflammation, respectively.

Dihydroxynonene mercapturic acid, a urinary metabolite of 4-hydroxynonenal, as a biomarker of lipid peroxidation.

The objective of our study was to compare the information obtained through the use of three different urinary biomarkers of lipoperoxidation during the time course of a bromotrichloromethane (BrCCl3) induced oxidative stress in rats. These biomarkers were malondialdehyde (MDA) measured by LC/MS after derivatization, the isoprostane 8-iso-PGF2alpha measured by enzyme immunoassay and 1,4-dihydroxynonene mercapturic acid (DHN-MA), the major 4-hydroxynonenal urinary metabolite [1], measured by LC-MS. Male Wistar rats received a single dose of 100 microL/kg BrCCl3 per os and lipid peroxidation was estimated every day for a 4-day-period after treatment. MDA, 8-iso-PGF2alpha and DHN-MA significantly increased in response to BrCCl3 treatment for this period of time, and DHN-MA showed the main increase during the 24-48 h period after treatment.

Increased proteolysis after single-dose exposure with hepatotoxins in HepG2 cells.

Chronic ethanol consumption is associated with increased protein oxidation and decreased proteolysis in the liver. We tested the hypothesis that even single-dose treatment with ethanol or bromotrichloromethane causes increased protein oxidation and a distinct proteolytic response in cultured hepatocytes. HepG2 cells were treated for 30 min with ethanol, H(2)O(2) and bromotrichloromethane at various nontoxic concentrations. Protein degradation was measured in living cells using [35S]-methionine labeling. Protein oxidation, and 20S proteasome activity were measured in cell lysates. Oxidized proteins increased immediately after ethanol, H(2)O(2), and bromotrichloromethane exposure, but a further significant increase 24-h after exposure was observed only following ethanol and bromotrichloromethane treatment. All three reagents caused a significant increase of the overall intracellular proteolysis at rather low concentrations, which could be suppressed by the proteasome inhibitor lactacystin. A decline of proteolysis observed at higher-subtoxic-concentrations was not related to decreased proteasome activity. Preincubation with ketoconazole or 4-methylpyrazole completely prevented the ethanol- and bromotrichloromethane-induced but not the H(2)O(2)-induced protein oxidation and proteolysis, suggesting strongly an enzyme-mediated generation of reactive oxygen species. In conclusion single-dose exposure with ethanol or haloalkanes causes increased protein oxidation followed by an increased proteasome-dependent protein degradation in human liver cells.

Use of rat liver slices for the study of oxidative DNA damage in comparison with isolated rat liver nuclei and HepG2 human hepatoma cells.

Tissue slices are a useful biological system for lipid peroxidation studies but their use for DNA damage studies is not well characterized. Hence, the present study investigates DNA damage in rat liver slices, in comparison with isolated rat liver nuclei and HepG2 human hepatoma cells, incubated with ferric nitrilotriacetate (Fe(III)-NTA), bromotrichloromethane (BrCCl(3)), bromobenzene (BrB) or 2-nitropropane (2-NP) at 37 degrees C for 2 hr. DNA damage was measured in slices, cells or nuclei after centrifugation as formation of as 8-hydroxy-2'-deoxyguanosine (8-OH-dGu) and loss of double-stranded (dsDNA) due to strand breakage using a fluorometric analysis of DNA unwinding (FADU). Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) released into the medium. The results show that in liver slices and isolated nuclei, Fe/NTA (1 mM/4 mM) induced high levels of TBARS but low levels of 8-OH-dGu, whereas the oxidant induced low levels of TBARS and no formation of 8-OH-dGu in HepG2 cells. In all three systems, inclusion of ascorbate caused dose-dependent formation of 8-OH-dGu, and the levels were similar between liver slices and HepG2 cells but were far higher in isolated nuclei. In liver slices the FADU assay was not applicable due to limited solubilization of DNA from the slice, whereas the assay detected significant loss of dsDNA in HepG2 cells and slight loss in isolated nuclei induced by Fe/NTA with or without ascorbate. Liver slices incubated with 1 mm BrCCl(3), BrB or 2-NP had elevated TBARS but had little or no formation of 8-OH-dGu; none of these oxidants induced lipid peroxidation or DNA damage in HepG2 cells. When liver slices obtained from rats injected with diethylmaleate (to deplete GSH) were incubated with BrCCl(3), BrB or 2-NP, levels of TBARS and 8-OH-dGu increased markedly. Similarly, HepG2 cells with decreased GSH showed marked elevation of TBARS and loss of dsDNA induced by these oxidants, although no formation of 8-OH-dGu was detected. The present study demonstrates the usefulness and limitations of liver slices for DNA damage studies and the importance of cellular GSH in the protection of DNA against environmental toxicants.

Degradation of chemicals by reactive radicals produced by cellobiose dehydrogenase from Phanerochaete chrysosporium.

Phanerochaete chrysosporium, grown on cellulose, produced a cellobiose-dependent dehydrogenase which reduced both ferric iron and molecular oxygen, resulting in the generation of the hydroxyl radical. The hydroxyl radical was detected in reaction mixtures with and without the addition of exogenous H2O2. The purified reductase and the fungus grown under nonligninolytic conditions that promote the production of the reductase were able to depolymerize an insoluble polyacrylate polymer. When oxalate, a secondary metabolite of P. chrysosporium, was used as the iron chelator, it was oxidized by the hydroxyl radical to form the carboxylate anion radical, a strong reductant. Under these reductive conditions, the enzyme was shown to catalyze the reduction of bromotrichloromethane to the trichloromethyl radical. We propose that these oxidative and reductive mechanisms may contribute to the degradation of a wide range of environmental pollutants by fungi which produce this enzyme.

Heme oxygenase active-site residues identified by heme-protein cross-linking during reduction of CBrCl3.

The reduction of CBrCl3 by the heme-heme oxygenase complex forms dissociable and covalently bound heme products. No such products are formed with mesoheme in which the heme vinyl substituents are replaced by ethyl groups. The dissociable heme products are chromatographically similar but not identical to those obtained in the analogous reaction with myoglobin. Tryptic digestion of the heme-protein adduct and Edman sequencing and mass spectrometric analysis of the heme-linked peptide identify His-25, the proximal iron ligand, as the alkylated residue. Reaction of CBrCl3 with the heme complexes of the T135V mutant and a Delta221 C-terminal truncated protein yields heme-linked peptides in addition to that from the wild-type reaction. The sequence of the principal labeled peptide from the T135V reaction, 205TAFLLNIQLFEELQELLTHDTK226 , and the lability of the adduct suggest the heme is attached to one of the carboxylic acid residues. A carboxylic acid residue is probably also labeled in the modified peptide 49LVMASLYHIYVALEEEIER67 from the Delta221 truncated protein. Thus, addition of the reductively generated trichloromethyl radical to a heme vinyl group produces a species that alkylates active-site residues. The changes in the alkylated residue caused by the Thr-135 mutation or truncation of the protein places residues in the sequences 49-67 and 205-226 within the active site. Furthermore, this is the first demonstration that heme oxygenase, like cytochrome P450, may catalyze the reductive metabolism of halocarbons and thus contribute to the toxicity of these agents.

Cholesterol interaction with free radicals produced from carbon tetrachloride or bromotrichloromethane by either catalytic decomposition or via liver microsomal activation.

The reaction between cholesterol (Ch) and trichloromethyl or trichloromethyl peroxyl radicals was studied. The latter were generated from CCl4 either by benzoyl peroxide (BP) catalysis or via thermal activation or by liver microsomal NADPH-dependent biotransformation of CBrCl3. The structure of the products formed was elucidated by gas chromatography-mass spectrometry (GC/MS). Under aerobic conditions and using thermal activation of CCl4, the formation of 6 products was observed. Two (I and II) were dehydrated Ch derivatives (one also having a third double bond) (I). Another product was a delta(5)-3 ketone derivative of Ch (III). Two additional reaction products were determined as ketocholesterols (IV and V). One chloro Ch was also formed (VI). At low concentrations of BP, reaction was more extensive than under thermal activation, and the formation of peaks I to IV was also observed. When the reaction was conducted anaerobically and using thermal activation of CCl4 to generate radicals, only products I and II were formed in low yield. Under anaerobic conditions, but using catalyst, compounds I and III were produced plus two new isomeric ketocholesterol derivatives (VIII and IX) and also a compound having an extra hydroxyl group on the Ch structure (X). In order to check whether similar reactions are observable under biological experimental conditions, we used activation of CBrCl3 by liver microsomes. The incubation using only microsomes (without CBrCl3 or NADPH) showed two ketocholesterol peaks (A and B). In the presence of CBrCl3 we could detect peak B and hydroxycholesterol (C) and two others, ketocholesterols (D and E). D was the only peak showing close similarity (spectrum and retention time) to one of those observed in the chemical reaction system (V). The reaction of CBrCl3 in the presence of NADPH showed peaks B, C, D and E, in low abundance and a 7-ketocholesterol (F). If some of the reaction products reported here were formed during the intoxication with these haloalkanes, significant biological consequences might be expected.

Reaction of bromotrichloromethane derived free radicals with uracil in a model system. Structures of products formed.

Free radicals generated by benzoyl peroxide-mediated catalytic decomposition of bromotrichloromethane (eg. trichloromethyl) were allowed to react under nitrogen or under air with uracil. Under nitrogen two reaction products were formed, one was identified as 5-chlorouracil and the other as a 5-bromouracil. Under air, besides the above two products other nine were also formed: 5,6-dihydrouracil; 5-hydroxyuracil; a chlorohydroxy adduct of uracil; a bromohydroxy derivative of uracil having the 5,6 bond in the saturated form; other bromohydroxy derivative of uracil having the double bond intact; 5,6-dihydroxyuracil; two dihalogenated hydroxylated uracil derivatives and one peak we were not able to descipher its structure. No single reaction product formed had carbon centered radicals (eg. trichloromethyl) added from CBrCl3 and consequently would be missed in 'in vivo' covalent binding studies where 14C haloalkane (CBrCl3 or carbon tetrachloride) were employed. If similar reaction products resulted during interaction of CBrCl3 reactive metabolites with uracil in RNAs, significant deleterious effects in their function would be expected. That possibility, however, remains to be established.

Suicidal inactivation of haemoproteins by reductive metabolites of halomethanes: a structure-activity relationship study.

Human haemoglobin (Hb), methaemalbumin (MHA) or rat liver microsomal cytochrome P-450 (P-450) were incubated anaerobically at microM concentrations with 1 mM carbon tetrachloride (CCl4), trichlorobromomethane (CCl3Br), chloroform (CHCl3) or methylene chloride (CH2Cl2) in presence of 1 mM sodium dithionite as the reducing agent. At the end of a 5-min incubation, haem was measured by various methods, i.e. binding spectrum with CO, pyridine-haemochromogen haem assay and porphyrin fluorescence, and compared for the four analogues. Statistically significant losses were observed, with all three haemo-protein systems, for CCi3Br, CCl4 and CHCl3, but not CH2Cl2. For Hb, the loss was greater with CCl3Br (haem assay, 63%; porphyrin fluorescence, 48%; CO binding, 24%) than with CCl4 (haem assay, 31%) or CHCl3 (haem assay, 13%). On the other hand, with MHA, CCl4 gave a dramatic loss (haem assay, 88%; porphyrin fluorescence, 83%; CO binding, 67%), which was greater than that observed with CCl3Br (haem assay, 49%; porphyrin fluorescence, 38%; CO binding, 25%). No loss was found with CHCl3. Finally, with microsomes, the inactivation was larger with CCl4 (CO binding, 58%; haem assay, 50%; porphyrin fluorescence, 33%) than with CCl3Br (CO binding, 33%; haem assay, 10%) or CHCl3 (haem assay, 9%; CO binding, 6%). In a separate set of similar experiments, an ion-pairing reverse phase HPLC method showed the formation of substrate-dependent hae-derived products during incubation of CCl3Br with Hb or microsomes, and of CCl4 with Hb. A correlation between potential for free radical formation (CCl3Br > CCl4 > CHCl3 > CH2Cl2) and extent of haem inactivation was observed with all methods for Hb, but not for microsomal P-450 or MHA. The results indicate that these halomethanes may be activated differently by different haemoproteins and suggest that their potential ability to undergo reductive metabolism may not be the only critical factor involved in P-450 haem inactivation by these chemicals.

5-methylcytosine attack by free radicals arising from bromotrichloromethane in a model system: structures of reaction products.

The interaction between free radicals derived from the catalytic decomposition of bromotrichloromethane and 5-methylcytosine (5MC) under different conditions were studied. The structures of the reaction products formed was established by the GC/MS analysis of their trimethylsilyl derivatives. Under anaerobic conditions, the formation of the following products was found: (1) thymine; (2) 5-hydroxymethyl uracil. Under aerobic conditions, the following reaction products were identified: (1) The same two products formed under anerobic conditions. (2) Monohydroxylated thymine. Precise location of the hydroxyl group was not established but probably corresponds to the six position isomer. (3) Two monochloro monohydroxy thymines. It is suggested that they are cis-trans isomers whose substituents are located at the 5-methyl and six positions of the base. (4) The trimethylsilyl derivative of thymine glycol. (5) Two monobromo monohydroxy adducts of thymine. One of them was detected as its underivatized form in the hydroxyl group position. (6) A partially silylated dihydroxythymine. When benzoyl peroxide was omitted from aerobic incubation mixtures, the compounds formed changed. No longer observable were: thymine; the two monochloro monohydroxy derivatives of thymine; thymine glycol, and one monohydroxythymine. On the other hand, two new reaction products were formed instead: a partially silylated monochloro-monohydroxy thymine and 5-hydroxymethyl-cytosine. If similar or equivalent reaction products were formed in DNA during CBrCl3 or CCl4 poisoning, results might be of relevance, because the 5MC content in DNA from eukaryotes is related to differentiation, gene control, and to carcinogenesis.

Potentiation of oxidative damage to rat red blood cells by the concurrent presence of t-butyl hydroperoxide and bromotrichloromethane.

Recently potentiation of oxidative damage in rat red blood cells (rRBC) incubated with t-butylhydroperoxide (BHP) in combination with bromotrichloromethane (BrCCI3) was demonstrated. The mechanism by which this combination (BrCCI3/BHP) potentiates the oxidative damage to rRBC was investigated in this study. When rRBC were incubated with 0.1 mM BHP, 0.5 mM BrCCI3, or the two combined, BrCCI3/BHP-potentiated lipid peroxidation and hemolysis were further enhanced under anaerobic conditions. However, the potentiation of lipid peroxidation was abolished by heating or trypsin digestion of rRBC. Electron spin resonance (ESR) studies demonstrated an increase of alkoyl radical induced by BrCCI3/BHP in rRBC, and this increase was abolished by heating or predigestion of hemolysates with trypsin. The inhibition of lipid peroxidation by diphenylamine (which reacts with alkoxyl radicals but not peroxyl radicals) suggests an important role of alkoxyl radicals. Overall, the present findings demonstrate that the increase in radical-related oxidative damage, possibly mediated by proteinlike materials, may be at least partially responsible for the potentiation of damage to rRBC induced by BrCCI3/BHP, and perhaps by BrCCI3. Although the in vivo significance of these results remains to be investigated, it seems likely that halocarbon toxicity may be amplified by elevated levels of lipid peroxide in blood.

The role of glutathione and protein thiols in CBrCl3-induced cytotoxicity in isolated rat hepatocytes.

The role of glutathione (GSH) and protein thiols in the pathobiochemical process of CBrCl3 cytotoxicity was investigated in isolated hepatocytes. Administration of 0.5, 1.0 and 1.5 mmol/l CBrCl3 affected cellular viability as assessed by trypan blue exclusion, release of lactate dehydrogenase and loss of intracellular potassium in a dose-dependent manner. Intracellular glutathione and the capacity to reduce 3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazolium bromide (MTT, thiazolyl blue) decreased almost independently of the CBrCl3 concentration. Protein thiols were not markedly oxidized in the presence of CBrCl3. However, compromising cellular defence mechanisms by either inhibition of glutathione regeneration or depletion of glutathione enhanced the cytotoxicity of CBrCl3 and induced a loss of protein thiols in the late phase of cellular injury. Under these conditions the thiol-dependent Na+,K+ATPase revealed high sensitivity towards CBrCl3. Thus, glutathione proved to exert effective cytoprotection, and sulfhydryl groups of particular proteins were supposed to be an important target of radical attack.

Structure of the novel heme adduct formed during the reaction of human hemoglobin with BrCCl3 in red cell lysates.

It was previously shown that the reductive debromination of BrCCl3 to trichloromethyl radical by human hemoglobin leads to formation of dissociable altered heme products, two of which are identical to those formed from myoglobin and one which is novel. In this study, we have elucidated the structure of this novel adduct with the use of mass spectrometry, as well as 1H and 13C NMR as a substitution product of a -C(Cl) = CCl2 moiety for a beta-hydrogen atom on the prosthetic heme's ring I vinyl group. From studies with the use of 13C-enriched BrCCl3, it was determined that the added carbon atoms were derived from 2 eq of BrCCl3. A mechanism that involves multiple reductive events and a radical cation heme intermediate is proposed. Consistent with this mechanism, cellular reductants were found to selectively enhance the amount of this novel dissociable heme adduct. These studies reveal fine differences between myoglobin and hemoglobin in the accessibility of reactive intermediates to the ring I vinyl group, as well as the potential importance of cellular reductants on the course of heme alteration.

The interactive toxicity of CHCl3 and BrCCl3 in precision-cut rat liver slices.

The interactive toxicity of two nontoxic concentrations of chloroform (CHCl3) and bromotrichloromethane (BrCCl3) was examined in precision-cut rat liver slices. Liver slices were prepared from male Sprague-Dawley rats (220-250 g) pretreated with phenobarbital for 4 days. Toxicants were administered 1 hr apart. Intracellular K+ levels were similar to untreated controls in slices treated with 0.2 mM CHCl3 or 0.125 microliters (0.25 mg, 1.26 mumol) BrCCl3 alone, indicating that these concentrations were nontoxic. However, addition of both toxicants, irrespective of order, resulted in a time-dependent loss of intracellular K+ which was significant at 9 hr following administration. This was interpreted as evidence of synergistic toxicity. Cytochrome P450 loss was significant as early as 3 hr following exposure to BrCCl3, alone or when added with CHCl3. This loss may be attributed to BrCCl3-induced suicide inactivation of cytochrome P450. Centrilobular hepatocytes may be more susceptible to the interactive toxicity of CHCl3 and BrCCl3. Activity of enzymes found predominantly in this area was significantly decreased in slices exposed to both toxicants relative to controls. Conversely, activity of enzymes found predominantly in the periportal region was similar to that of untreated and treated controls. Interactive toxicity of BrCCl3 and CHCl3 was not a consequence of increased lipid peroxidation or depletion of slice glutathione content. Further studies need to be conducted to elucidate the mechanisms mediating the interactive toxicity of BrCCl3 and CHCl3.

Trichlorobromomethane-induced changes of purine nucleotides in hepatocytes.

The changes in nucleotide content during CBrCl3 treatment were investigated. In the first 5-10 minutes a significant ATP decrease was detected. The GTP loss leading to 57% of the initial level after 10 min surpasses the ATP loss which leads to 70% of the initial value after 10 min. The increase in uric acid is not only the result of CBrCl3 induced reaction, because of no significant changes in adenine and hypoxanthine values. The uric acid pool reflected different influx and efflux processes. These changes were compared with nucleotide degradation and accumulation of nucleotide degradation products during anoxia.

Oxidation of heme proteins as a measure of oxidative damage to liver tissue slices.

Oxidative damage to heme proteins in rat liver tissue slices was studied. Tissue slices were incubated in Krebs-Ringer phosphate (KRP) buffer at 37 degrees C with and without the presence of prooxidants. The absorbance spectra (500-640 nm) of heme proteins of tissue slices obtained from both spontaneous and prooxidant-induced oxidation were analyzed with a heme protein spectra analysis program (HPSAP) developed in this laboratory. The dominant heme proteins in a fresh nonperfused tissue slice were hemoglobin and reduced cytochromes of mitochondria. In an oxidized tissue slice, the major oxidized product was hemichrome. Bromotrichloromethane, t-butyl hydroperoxide, and ferrous ion accelerated the oxidative reactions, and the amount of oxidized products was dependent on the incubation time as well as the type and concentration of prooxidants.

Metabolism-based transformation of myoglobin to an oxidase by BrCCl3 and molecular modeling of the oxidase form.

The stoichiometric reductive debromination of BrCCl3 to a trichloromethyl radical by myoglobin caused the prosthetic heme to become covalently cross-linked to the protein moiety and transformed myoglobin from an oxygen storage protein to an oxidase. This was shown in experiments in which oxygen consumption was measured during redox cycling of the altered myoglobin in the presence of ascorbate or an enzymatic reducing system containing diaphorase and NADH. Redox cycling eventually led to loss of the protein-bound heme adduct and oxidase activity of myoglobin. We have used molecular modeling and the known structure of the protein-bound heme adduct to identify probable mechanisms for transformation of myoglobin to an oxidase. Based on these modeling studies, the most likely structure of the experimentally observed adduct involves ligation to the heme iron of the epsilon-nitrogen atom of histidine 97 and/or that of histidine 64. The model structures revealed access of solvent to the heme active site, which could facilitate oxygen reduction. The transformation of myoglobins and perhaps other hemoproteins to oxidases may have toxicological importance in causing the tissue damage resulting from exposure to various xenobiotics and endogenous chemicals as well as explaining how hemoproteins are inactivated during catalysis.

Determination of 4-hydroxynonenal by high-performance liquid chromatography with electrochemical detection.

4-Hydroxy-trans-2-nonenal (HNE) is a highly reactive product of lipid peroxidation originating from the break-down of phospholipid-bound polyunsaturated fatty acids of cellular membranes. Despite its biological relevance, this aldehyde is only occasionally determined due to the complexity of previously described procedures. Here we present a simple and very sensitive method for the detection of HNE in biological samples. The method is based on the measurement of the 2,4-dinitrophenylhydrazone (DNPH) of the aldehyde by electrochemical detection after separation by reverse-phase high-performance liquid chromatography (HPLC). The greater sensitivity of this procedure as compared to the ultraviolet detection method commonly employed to measure DNPH derivatives of aldehydes after HPLC will allow the detection of HNE below the pmol level. The detection of HNE is highly reproducible even in normal tissues and cells. Increased amounts of HNE were detected in the livers of animals intoxicated with prooxidant agents such as carbon tetrachloride, bromotrichloromethane or bromobenzene. An exponential increase in HNE (and in malondialdehyde) was measured in peroxidizing liver microsomes (in the NADPH/Fe-dependent system). The method is also suitable for the study of very small samples, since HNE could be detected in approximately 1 million cultured cells (polyoma virus-transformed baby hamster kidney fibroblasts); the level rose after exposure of the cells to a Fe3+/ADP prooxidant system.

Cytosine attack by free radicals arising from bromotrichloromethane in the presence of benzoyl peroxide catalyst: a mass spectrometric study.

We and others previously reported that CCl4 reactive metabolites are able to covalently bind to liver DNA either in vivo or in vitro. However, no demonstration of the structure of resulting adducts is available in literature. That information would be of relevance, for CCl4 exhibits null or contradictory mutagenic properties and is currently considered a non-genotoxic carcinogen. In the present study we report the nature of the reaction products formed when the putative CCl4 metabolites, .CCl3 and CCl3O2. attack cytosine in a purely chemical system where they were generated from CCl3Br in a benzoyl peroxide catalyzed reaction. Reaction products formed and identified were a) under nitrogen (.CCl3 present)--5-bromo cytosine and cytosine-5-carboxylic acid; b) under air (CCl3O2. present)--5-bromo cytosine, 5-chloro cytosine, 5-hydroxy cytosine, 6-hydroxy cytosine (tentative), chloro hydroxy uracil, 5,6-dihydroxy uracil, and chloro trichloromethyl cytosine. Results from present experiments suggest that if these reaction products were also produced in vivo during either CCl4 or CCl3Br poisoning and they were not repaired in due time prior to replication, they would lead to mutagenic events. Studies directed to obtain evidence for their in vivo formation are in course in our laboratory.

In vivo studies on halogen compound interactions. IV. Interaction among different halogen derivatives with and without synergistic action on liver toxicity.

The liver toxicity of several halogen compound mixtures have been tested. The compounds were selected on the basis of their metabolic pathways: carbon tetrachloride (CT) and trichlorobromomethane (TCBM) undergo a dehalogenation via P450-dependent enzyme system, 1,2-dichloroethane (DCE) and 1,2-dibromoethane (DBE) are mainly conjugated with the cytosolic glutathione (GSH) by means of the GSH-S-transferase. The mixture TCBM+DBE shows a more than additive action on lipid peroxidation and liver necrosis. TCBM, like CT, reduces the hepatic level of GSH-S-transferase, increasing the amount of DBE available for cytochrome P450-dependent metabolism, with the production of toxic metabolites. Thus, the behavior of the mixture TCBM+DBE is very similar to that of the mixture CT+DBE, previously reported. Mixtures composed of CT+TCBM and DCE+DBE do not show any synergistic effect on liver toxicity. The results allow one to conclude that the toxicity of mixtures of halogen compounds can be partly predicted on the basis of their metabolic pathways. When the metabolism is quite different, a synergistic toxicity can occur if one pathway interferes with a detoxification mechanism of the other compound. If the two metabolisms are very similar they produce, at most, an additive toxicity.