Tamoxifen metabolic activation: comparison of DNA adducts formed by microsomal and chemical activation of tamoxifen and 4-hydroxytamoxifen with DNA adducts formed in vivo.

One of our laboratories recently showed by 32P-postlabeling that administration of tamoxifen to mice induces two groups of hepatic DNA adducts comprising two major spots, nos. 3 and 5, respectively. 4-Hydroxytamoxifen and alpha-hydroxytamoxifen appear to be the proximate metabolites of groups I and II adducts, respectively. The relative significance of these two adduct groups for tamoxifen carcinogenicity remains to be established. To determine the activation mechanism(s) of tamoxifen and 4-hydroxytamoxifen, in vivo adducts were compared by 32P-postlabeling with adducts generated by microsomal or chemical activation in vitro. Microsomal activation of 4-hydroxytamoxifen and tamoxifen, respectively, in the presence of DNA and cumene hydroperoxide, induced two adducts, which mapped similarly to the corresponding in vivo adduct spots 3 and 5. Chemical oxidation of 4-hydroxytamoxifen with silver(II) oxide, followed by incubation of the product(s) with DNA, elicited the formation of a major spot (Q1), while tamoxifen itself did not react. Rechromatographic analyses revealed that in vitro fractions 3 and Q1 (from 4-hydroxytamoxifen) matched the major in vivo group I adduct fraction 3, consistent with the hypothesis that 4-hydroxytamoxifen is a precursor for adduct fraction 3 in vivo. The in vitro adduct fraction 5 (from tamoxifen) was identical to that formed in vivo, indicating that the metabolic pathway for the formation of group II adducts did not involve 4-hydroxytamoxifen. In conclusion, the results support a model where primary metabolites of tamoxifen undergo secondary metabolism to form DNA adducts, which are detected in vivo after treatment with tamoxifen or 4-hydroxytamoxifen.

Activation of the tamoxifen derivative metabolite E to form DNA adducts: comparison with the adducts formed by microsomal activation of tamoxifen.

(Z)-1,2-Diphenyl-1-(4-hydroxyphenyl)but-1-ene (metabolite E) has been detected in the plasma of patients treated with tamoxifen. We therefore investigated whether the cis/trans isomers of metabolite E can be activated to form DNA adducts detected by 32P postlabeling. Microsomal activation of metabolite E produced two major (a and b) and up to six minor DNA adducts. Activation with horseradish peroxidase or silver(I)oxide produced the same adducts (a and b). Microsomal activation of tamoxifen produced one major (no. 6) and several minor DNA adducts. Rechromatography showed that adducts a and b formed by enzymatic and chemical activation of metabolite E were the same as adducts 9 and 4 produced by microsomal activation of tamoxifen. These results demonstrate that activation of metabolite E can lead to DNA adduct formation.

In vivo binding of diethylstilbestrol to nuclear proteins of kidneys of Syrian hamsters.

We demonstrate here that stilbene estrogen (diethylstilbestrol) is converted to nuclear protein binding metabolite(s) both in vitro and in vivo. In vitro reaction of DES with nuclei from hamster liver or kidney in the presence of cumene hydroperoxide or NADPH revealed binding of [3H]DES in nuclear proteins (histones; nonhistones precipitable by 2% TCA, NH2; nonhistones soluble in 2% TCA, NH30). The binding was significantly inhibited by cytochromes P450 inhibitors. In an in vitro system [3H]DES quinone, one of the metabolites of DES, was able to bind to pure nonhistone proteins RNA polymerase and DNA polymerase. The binding of [3H]DES quinone to nonhistones RNA polymerase and DNA polymerase was inhibited by low molecular weight thiols, i.e. glutathione and cysteine, or thiol modifiers, such as n-ethylmaleimide, dithionitrobenzoic acid and hydroxymercuric benzoate. DES and DES metabolites inhibited transcriptional activity. In vivo [3H]DES was able to bind to nuclear proteins of hamster liver, kidneys and testes. The level of in vivo [3H]DES binding to all three types of nuclear proteins (histones, NH2, NH30) in the kidney (target organ) was two or more fold higher than that observed in the liver or testis (nontarget organs). Four nuclear NH30 proteins (mol wts.: 56, 37, 33 and 28 kDa) were irreversibly bound to [3H]DES in vivo. The in vivo binding of [3H]DES to transcriptionally active chromatin NH30 proteins also was observed. The data reported here establish that DES was able to bind to liver or kidney nuclear proteins in vitro, which was catalyzed by nuclear enzymes when fortified with an appropriate cofactor. DES quinone may be one of the protein binding metabolites. DES and DES metabolites inhibited transcriptional activity. The level of in vivo binding of [3H] DES to nuclear proteins of kidney (target organ) was double in comparison with that observed in liver or testis (nontarget organs). In vivo modifications in the chromatin proteins may be a factor in the development of DES-induced renal carcinogenesis is not clear.

Effect of centrophenoxine on the antioxidative enzymes in various regions of the aging rat brain.

This study investigated the effect (in vivo) of centrophenoxine (Helfergin) on the activity of antioxidant enzymes (glutathione peroxidase GSH-PER, glutathione reductase GSSG-RED, superoxide dismutase SOD and catalase) in subcellular fractions from the regions of the brain (cerebrum, cerebellum and brain stem) of rats aged 6, 9 and 12 months. In all age groups, normal (control) activity of GSH-PER, GSSG-RED and SOD in the three brain regions was higher in the soluble fractions than in the particulate fractions. The three regions of the brain showed different levels of the enzyme activities. Enzymes in soluble fractions (except GSSG-RED in cerebrum of rats aged 12 months) did not change with age. In particulate fractions, however, the enzymes showed age-related changes: GSH-PER decreased with age in cerebellum and brain stem, but showed an age-related increase in cerebrum, GSSG-RED and SOD increased with age in all the three brain regions. Catalase activity in all the three brain regions remained unchanged in all age groups. Six week administration of centrophenoxine (once a day in doses of 80 mg/Kg and 120 mg/Kg) to the experimental animals produced increases in the activity of SOD, GSH-PER and GSSG-RED in particulate fractions from all the three brain regions. In the soluble fractions, however, only SOD and GSH-PER activity was increased. In vitro also centrophenoxine stimulated the activity of GSH-PER. A dosage of 80 mg/Kg produced greater changes than a 120 mg/Kg dosage. The drug had no effect on the activity of catalase. Centrophenoxine also reduced lipofuscin deposits (studied both biochemically and histochemically) thus indicating that the drug inhibited lipofuscin accumulation by elevating the activity of the antioxidant enzymes. The data suggest that alleviation of senescence by centrophenoxine may, at least, partly be due to activation by it of antioxidant enzymes.

Investigation of the DNA adducts formed in B6C3F1 mice treated with benzene: implications for molecular dosimetry.

We have investigated the formation of DNA adducts in the bone marrow and white blood cells of male B6C3F1 mice treated with benzene using P1-enhanced 32P-postlabeling. No adducts were detected in the bone marrow of controls or mice treated with various doses of benzene once a day. After twice-daily treatment for 1 to 7 days with benzene, 440 mg/kg, one major (no. 1) and up to two minor DNA adducts were detected in both the bone marrow and white blood cells. The relative adduct levels in these cells ranged from 0.06 to 1.46 x 10(-7). a significant correlation (r2 = 0.95) between levels of adducts in bone marrow and white blood cells was observed. After a 7-day treatment with benzene, 440 mg/kg twice a day, the number of cells per femur decreased from 1.6 x 10(7) to 0.85 x 10(7), indicating myelotoxicity. In contrast, administration of benzene once a day produced only a small decrease in bone marrow cellularity. The observed induction of toxicity in bone marrow was paralleled by formation of DNA adducts. In vitro treatment of bone marrow with hydroquinone (HQ) for 24 hr produced the same DNA adducts as found after treatment of mice with benzene, suggesting that HQ is the principal metabolite of benzene leading to DNA adduct formation in vivo. Using P-postlabeling the principal DNA adduct formed in vivo was compared with N2-(4-hydroxyphenyl)-2'-deoxyguanosine-3'-phosphate. The results of this comparison demonstrated that the DNA adduct formed in vivo co-chromatographs with N2-(4-hydroxyphenyl)-2'-deoxyguanosine-3'-phosphate. These studies indicate that metabolic activation of benzene leads to the formation of DNA adducts in bone marrow and white blood cells and suggest that measurement of DNA adducts in white blood cells may be an indicator of biological effect following benzene exposure.

In vivo genotoxicity of sodium ortho-phenylphenol: phenylbenzoquinone is one of the DNA-binding metabolite(s) of sodium ortho-phenylphenol.

We have previously demonstrated microsomal cytochromes P450-dependent redox cycling of o-phenylphenol and in vitro genotoxicity of o-phenylphenol. In the present work, we have investigated in vivo covalent modification in skin DNA by Na-o-phenylphenol using the 32P-postlabeling method in an attempt to understand the biochemical mechanism of promotion of chemical-induced skin carcinogenesis by Na-o-phenylphenol. Topical application of Na-o-phenylphenol or phenylhydroquinone, a hydroxylated metabolite of o-phenylphenol, to female CD-1 mice skin produced 4 distinct major and several minor adducts in skin DNA. The total covalent bindings in skin DNA produced by treatment of mice with 10 mg and 20 mg Na-o-phenylphenol (doses shown to be effective for tumor promotion) were 0.31 fmoles/microgram DNA and 0.62 fmoles/microgram DNA, respectively. The adducts were not observed in untreated animal skin DNA. Pretreatment of mice with alpha-naphthylisothiocyanate, an inhibitor of cytochromes P450, or indomethacin, an inhibitor of prostaglandin synthase, resulted in lower levels of DNA adducts produced by Na-OPP. The in vitro incubation of DNA with o-phenylphenol or phenylhydroquinone in the presence of cytochromes P450 activation or prostaglandin synthase activation system produced 4 major adducts. The adduct pattern observed in the presence of in vitro enzymatic activation systems appears to be similar in chromatographic mobility to the in vivo adduct pattern. The chemical reaction of DNA or deoxyguanosine monophosphate with pure phenylbenzoquinone, an electrophilic metabolite of o-phenylphenol, also produced 4 major and several minor adducts. The 4 major adducts obtained in chemical reaction of phenylbenzoquinone with deoxyguanosine monophosphate are identical in chromatographic mobility to those of in vivo or in vitro DNA adducts. The results of this study demonstrated that o-phenylphenol or phenylhydroquinone, a hydroxylated metabolite of o-phenylphenol, is able to covalently bind to DNA. DNA binding can be inhibited by the inhibitor of cytochromes, P450 alpha-naphthylisothiocyanate or prostaglandin synthase, indomethacin. One of the DNA-binding metabolite(s) of o-phenylphenol both in vivo and in vitro may be phenylbenzoquinone. We conclude that Na-OPP is genotoxic. Genotoxicity caused by Na-o-phenylphenol treatment in CD-1 mice may play a role in the promotion of dimethylbenz[a]anthracene-induced skin neoplasm.

Histone nuclear proteins are irreversibly modified by reactive metabolites of diethylstilbestrol.

We demonstrate for the first time that diethylstilbestrol (DES), a synthetic estrogen, is converted by nuclei to histone-binding metabolite(s). Reaction of [3H]DES with nuclei in the presence of cumene hydroperoxide or NADPH revealed binding of [3H]DES to histone nuclear proteins. Gel electrophoresis experiments revealed that all five histones, 1, 2A, 2B, 3, and 4, were irreversibly bound to [3H]DES. Histones 1 and 3 were more susceptible to the attack by [3H]DES quinone, a metabolite of DES, than histones 2A, 2B, or 4. The kinetic constants, Km and Vmax, of this binding reaction in the presence of cumene hydroperoxide were 10 microM and 750 pmol/mg protein/30 min, respectively. This binding was significantly inhibited by cytochromes P-450 inhibitors. Low-molecular-weight thiols, such as glutathione and cysteine, or thiol modifiers, such as n-ethylmaleimide, dithionitrobenzoic acid, and hydroxymercuric benzoate, drastically inhibited binding of [3H]DES quinone to histone 3. The binding of [3H]DES metabolites to both transcriptionally active and inactive chromatin histone proteins was observed. We conclude that DES is metabolized to histone-binding metabolites, presumably by nuclear cytochrome P-450. DES quinone may be one of the histone-binding DES metabolites. These data suggest that an analogous in vivo modification in the transcriptionally active chromatin histones by DES metabolites may influence gene function.

DNA adduct formation by tamoxifen with rat and human liver microsomal activation systems.

Using microsomal preparations from rat and human liver, we investigated the activation of the anti-estrogen compound tamoxifen (TMX) to form DNA adducts. Pretreatment of rats with phenobarbital increased DNA adduct formation by microsomal activation of TMX 3- to 6-fold, depending on the cofactors used. When reduced nicotinamide-adenine dinucleotide phosphate (NADPH) was used as a cofactor in human and rat microsomal activation systems, the relative DNA adduct levels were 2.9 and 5.2 x 10(-8) respectively and 1-3 TMX-DNA adducts were detected by 32P-postlabeling; DNA adduct 1 was the same in both microsomal systems. When cumene hydroperoxide (CuOOH) was used as a cofactor, activation of TMX produced four major DNA adducts and several minor DNA adducts in both rat and human liver microsomes; the relative adduct levels were 11.1 and 23.1 x 10(-8) respectively. TMX-DNA adducts 1, 4, 5 and 6 were similar in both human and rat microsomal systems with CuOOH as the cofactor. The TMX-DNA adducts formed with NADPH as the cofactor were clearly different from those formed with CuOOH as the cofactor, which implies that the metabolites leading to the individual DNA adducts were different. Addition of a P450 inhibitor, either n-octylamine or alpha-naphthylisothiocyanate, to the activation system reduced adduct formation by 70-93%. We propose that the TMX-DNA adducts formed with NADPH as the cofactor result from P450 acting as a mono-oxygenase, whereas the adducts formed with CuOOH as the cofactor result from P450 acting as a peroxidase. Our findings suggest that further studies may be required to establish the safety of TMX treatment of women for purposes other than chemotherapy.

Modifications in the low mobility group nuclear proteins by reactive metabolites of diethylstilbestrol.

In this study we have investigated the potential of nuclear activation system to convert diethylstilbestrol (DES) to reactive metabolites, which bind to low mobility nonhistone nuclear proteins. Reaction of DES with nuclei in the presence of cumene hydroperoxide or NADPH revealed binding of DES in low mobility group (LMG) nonhistone nuclear proteins analyzed by both organic solvent extraction and gel electrophoresis methods. Gel electrophoresis experiments revealed that five LMG proteins of MW 130, 108, 72, 51, and 45 KDa were irreversibly bound to 3H-DES. The kinetic constants, Km and Vmax, of this binding reaction in the presence of cumene hydroperoxide were 39 uM and 1225 pmol/mg protein/30 min, respectively. This binding was significantly inhibited by cytochromes P450 inhibitors. Low molecular weight thiols, i.e., glutathione and cysteine, or thiol modifiers such as n-ethylmaleimide, dithionitrobenzoic acid, and hydroxymercuric benzoate, drastically inhibited binding. The binding of DES metabolites to both transcriptionally active and inactive chromatins LMG proteins was observed. In summary, DES is metabolized to transcriptionally active chromatin LMG protein binding metabolites presumably by nuclear cytochromes P450. These data suggest that an analogous in vivo modification in the transcriptionally active chromatin LMG nonhistone proteins by DES metabolites may influence gene transcription.

Examination of microsomal cytochrome P450-catalyzed in vitro activation of o-phenylphenol to DNA binding metabolite(s) by 32P-postlabeling technique.

It has been previously reported that the reactive metabolites phenylsemiquinone and phenylbenzoquinone are generated during microsomal cytochrome P450-catalyzed redox cycling of o-phenylphenol (OPP). However, covalent modification of DNA by OPP-reactive metabolites has yet not been demonstrated. In the present study we have investigated the covalent binding in DNA by OPP-reactive metabolites using 32P-postlabeling. Analysis of adducts by 32P-postlabeling in products of chemical reaction of DNA with phenylbenzoquinone revealed four major and several minor adducts. The chemical reaction of deoxyguanosine 3'-phosphate with phenylbenzoquinone also showed four major adducts. The chromatographic mobility of major adducts of deoxyguanosine 3'-phosphate-phenylbenzoquinone was identical to that of major adducts of DNA-phenylbenzoquinone. The major adducts are demonstrated to be stable. The total covalent binding in deoxyguanosine 3'-phosphate by phenylbenzoquinone (686,000-687,000 amol/nmol nucleotide) was higher than that observed in DNA (26,500-28,000 amol/nmol nucleotides). Reaction of DNA with OPP or a hydroxylated metabolite of OPP, phenylhydroquinone, in the presence of microsomes and NADPH or cumene hydroperoxide showed four major adducts. Adduct formation in DNA by OPP or phenylhydroquinone in the presence of the microsomal activation system was drastically decreased by known inhibitors of cytochrome P450. The chromatographic mobility of major adducts in DNA by OPP or phenylhydroquinone in the presence of microsomal activation system matched with those major adducts observed in deoxyguanosine 3'-phosphate or DNA reacted with pure phenylbenzoquinone. These data demonstrate that OPP or phenylhydroquinone, a hydroxylated metabolite of OPP, is able to bind covalently to DNA in the presence of a microsomal cytochrome P450 activation system. Phenylbenzoquinone is one of the DNA-binding metabolite(s) of OPP. It is concluded that OPP is genotoxic in an in vitro system and genotoxicity produced by OPP-reactive metabolites may play a role in OPP-induced cellular toxicity or cancer.

Lipid peroxidation and glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activities in FeCl3-induced epileptogenic foci in the rat brain.

This study investigated the relationship between lipid peroxidation, subsequent activation of antioxidative enzymes, and development of iron-induced epilepsy in the rat. Epileptic foci were produced in rat cerebral cortex by intracortical injection of ferric chloride (FeCl3). The epileptic foci were identified by electrocorticography (ECoG). Epileptiform ECoG activity was shown to occur in the contralateral homotopic cerebral cortex as well. We measured levels of lipid peroxides and changes in the activities of the enzymes: superoxide dismutase (SOD), glutathione peroxidase (GP), glutathione reductase (GR), catalase (CA), and glucose-6-phosphate dehydrogenase (G6P) in the epileptogenic focus (both ipsilateral and contralateral) at days 3, 8, 15, and 23 after FeCl3 injection. Biochemical estimations were made in subcellular fractions, and changes in the ipsilateral site were compared with those in the contralateral site. The results of this study showed that large increases in lipid peroxidation were associated with development and buildup of the ECoG epileptiform discharges. Lipid peroxides increased in the ipsilateral focus by approximately 100% as compared with control. In the contralateral site, however, the increase in lipid peroxides was marginal only. The increase in lipid peroxidation was concomitant with development of the high level of epileptiform activity. The time course of changes in lipid peroxidation paralleled the time course of development and persistence of the epileptiform activity. Regarding changes in the enzyme activities accompanying development of iron epilepsy, the data showed that although SOD and G6P increased by approximately 60% and GR increased by approximately 40%, the increases in the enzyme GP and CA were much lower, less than 20%. Thus, comparatively less increase in CA and GP activities produces a deficiency of these two enzymes in the iron (ipsilateral) focus. Among the various biochemical disturbances that have been identified as involved in epileptogenesis, peroxidative injury resulting from lipid peroxidation in neural plasma membrane may be causally related to development of paroxysmal epileptiform activity in the iron focus. Since GP is an enzyme of major importance in detoxification of lipid peroxides in the brain, based on the results presented in this article, it appears reasonable to suggest that GP deficiency causes lipid peroxidation to increase tremendously during iron epileptogenesis.

Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen.

Using rat liver microsomal preparations and peroxidase enzymes, we have investigated the formation of DNA adducts by the antiestrogen compound tamoxifen (TAM) and its metabolite 4-hydroxy-tamoxifen (4-OH-TAM). When reduced nicotinamide-adenine dinucleotide phosphate (NADPH) was used as a cofactor in microsomal activation of either 4-OH-TAM or TAM, one DNA adduct and relative DNA adduct levels of 4.6 and 3.1 x 10(-8), respectively were detected by 32P-postlabeling. The DNA adduct produced by microsomal activation of 4-OH-TAM and TAM was the same. With cumene hydroperoxide (CuOOH) as the cofactor for the microsomal activation of either 4-OH-TAM or TAM, three to six DNA adducts were produced; the relative adduct levels were 8.0 and 20.6 x 10(-8), respectively. Comparison of the DNA adduct patterns produced by 4-OH-TAM and TAM showed that they were distinct. However one of the DNA adducts (a) produced by microsomal activation of 4-OH-TAM using CuOOH was the same as adduct a produced by microsomal activation of 4-OH-TAM with NADPH. Activation of 4-OH-TAM with horseradish peroxidase resulted in the formation of a single DNA adduct and a relative adduct level of 20.7 x 10(-8). Rechromatography analysis of this DNA adduct showed that it was identical to that produced by microsomal activation of 4-OH-TAM with NADPH and one of the adducts produced using CuOOH as the cofactor. Ten DNA adducts and a relative adduct level of 15.3 x 10(-8) were detected in the liver of female Sprague-Dawley rats treated daily with 20 mg/kg of TAM for 7 days. The DNA adduct pattern in the liver of the treated animals was similar to that produced by microsomal activation of TAM using CuOOH as the co-factor. The principal DNA adduct (no. 6) formed in the livers of rats treated with TAM was the same as the principal DNA adduct formed following microsomal activation of TAM using CuOOH as a cofactor. The DNA adduct formed following microsomal activation of either TAM or 4-OH-TAM using NADPH was also present as one of the adducts (1) formed in vivo following TAM treatment. These studies demonstrate that 4-OH-TAM can be activated to form DNA adducts and that it contributes to the formation of DNA adducts in the liver of rats treated with TAM.

DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene.

We used P1-enhanced 32P-postlabeling to investigate DNA adduct formation in the bone marrow of B6C3F1 mice treated intraperitoneally with benzene (BZ). No adducts were detected in the bone marrow of controls or mice treated with various doses of BZ once a day. After twice-daily treatment with BZ, 440 mg/kg, for 1 to 7 days, one major and two minor DNA adducts were detected. The relative adduct levels ranged from 0.06-1.46 x 10(-7). In vitro treatment of bone marrow from B6C3F1 mice with various doses of hydroquinone (HQ) for 24 h also produced three DNA adducts. These adducts were the same as those formed after in vivo treatment of bone marrow with BZ. Co-chromatography experiments indicated that the principal DNA adduct detected in the bone marrow of B6C3F1 mice was the same as that detected in HL-60 cells treated with HQ. This finding suggests that HQ may be the principal metabolite of BZ leading to DNA adduct formation in vivo. DNA adduct 2 corresponds to the DNA adduct formed in HL-60 cells treated with 1,2,4-benzenetriol. DNA adduct 3 remains unidentified. After a 7-day treatment with BZ, 440 mg/kg twice a day, the number of cells per femur decreased from 1.6 x 10(7) to 0.85 x 10(7), indicating myelotoxicity. In contrast, administration of BZ once a day produced only a small decrease in bone marrow cellularity. These studies demonstrate that metabolic activation of BZ leads to the formation of DNA adducts in the bone marrow. Further investigation is required to determine the role of DNA adducts and other forms of DNA damage in the myelotoxic effects of exposure to BZ.

Activation of 4-hydroxytamoxifen and the tamoxifen derivative metabolite E by uterine peroxidase to form DNA adducts: comparison with DNA adducts formed in the uterus of Sprague-Dawley rats treated with tamoxifen.

Daily intraperitoneal treatment of female Sprague-Dawley rats with either 5, 10 or 20 mg/kg tamoxifen (TAM) for 1 week increased the level of peroxidase activity in the uterus 2- to 10-fold compared to the control level. Using uterine extracts prepared from control and TAM treated animals, we investigated the activation of 4-hydroxytamoxifen (4-HO-TAM) and (E,Z)-1,2-diphenyl-1-(4-hydroxyphenyl)-but-1-ene (cis/trans-metabolite E) to form DNA adducts. Activation of 4-HO-TAM by uterine extracts prepared from either control or TAM-treated rats produced one major (a) and two minor DNA (b and c) adducts. A similar activation of cis/trans-metabolite E produced two adducts (d and e). There was good correlation between levels of uterine peroxidase activity and levels of DNA adducts formed by 4-HO-TAM and cis/trans-metabolite E. Activation of 4-HO-TAM and cis/trans-metabolite E with horseradish peroxidase (HRP) produced the same adducts as observed by activation with uterine extract. Treatment of Sprague-Dawley rats with 5 and 10 mg/kg for 7 days produced eleven DNA adducts in the liver with no adducts detected in the uterus. However, treatment of rats with 20 mg/kg of TAM for 7 days produced the same adduct pattern in the liver and also one major adduct (1) in the uterus with a relative adduct level of 6.4 - 4.1 x 10(-9). Tamoxifen-DNA adduct 1 detected both in the liver and in the uterus of treated rats was similar to adducts produced by activation of 4-HO-TAM with either uterine extract or HRP. The results of these studies suggest a general model whereby the tamoxifen metabolite 4-HO-TAM is further activated in the uterus by peroxidase enzymes to form DNA adducts.

Effects of chlorpromazine on the activities of antioxidant enzymes and lipid peroxidation in the various regions of aging rat brain.

In this work, the effect of chronic intraperitoneal administration of chlorpromazine (5 and 10 mg/kg) on the antioxidant enzymes superoxide dismutase (SOD), catalase (CA), glutathione reductase (GR), and glutathione peroxidase (GP); lipid peroxidation; and lipofuscin accumulation in the brains of rats ages 6, 9, and 12 months was studied. Chlorpromazine increased the activities of SOD, GR, and GP in particulate fraction from cerebrum, cerebellum, and brain stem in a dose-dependent manner. While GR and SOD associated with soluble fraction increased, GP associated with soluble fraction was not affected. CA did not change after chlorpromazine administration in any regions of the brain of rats from all age groups. Chlorpromazine, thus, had a somewhat different action on antioxidant enzymes in different subcellular fractions. Chlorpromazine inhibited lipid peroxidation, both in vivo and in vitro, and it also inhibited accumulation of lipid peroxidation fluorescent products (lipofuscin), which was studied histochemically and biochemically as well. The data indicate that chlorpromazine inhibition of lipid peroxidation and of accumulation of lipofuscin can result from elevation of the activity of brain antioxidant enzymes.

Changes in the activity of gamma-amino butyric acid transaminase and succinic semialdehyde dehydrogenase in the cobalt and iron experimental epileptogenic foci in the rat brain.

GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSA-DH) activities were measured in the mitochondrial fractions from the cobalt- and FeCl3-induced chronic epileptogenic foci in the rat brain. Electroencephalographically, the FeCl3 epileptogenic focus remained active for a duration longer than that of the cobalt focus. In both the foci SSA-DH activity showed significant increases which were concomitant with the EEG epileptiform activity. In cobalt focus, the GABA-T activity fell whereas, in the FeCl3 focus it was unchanged. In cobalt focus fall in GABA-T activity seemed to be concomitant with EEG epileptiform discharge. The measurements of the enzyme activities in the mirror (secondary) foci showed that, except for a brief stimulation of SSA-DH activity in the mirror focus in FeCl3 epileptic animals, the enzyme activities remained unchanged. Possible significance of the observed enzymatic changes in the physiology of epileptogenic focus is discussed.

Detection of DNA adducts in the white blood cells of B6C3F1 mice treated with benzene.

We have employed the P1-enhanced 32P-postlabeling procedure to detect the formation DNA of adducts in the white blood cells (WBC) of B6C3F1 mice treated by i.p. injection with benzene. Treatment twice a day with 440 mg/kg benzene for 1-7 days resulted in the formation of one major (adduct 1) and one minor (adduct 2) DNA adduct in the WBCs of mice. The same DNA adduct pattern was also found in the bone marrow (BM) of benzene treated mice. The relative adduct levels were dependent upon both benzene dose from 100-440 mg/kg and treatment time from 1 to 7 days. The relative adduct levels ranged between 0.11 and 1.33 adducts in 10(7) nucleotides for WBCs and 0.16-1.21 adducts in 10(7) nucleotides for BM. Following treatment with benzene, the levels of DNA adducts formed in WBCs were significantly correlated with the levels of DNA adducts formed in BM (r2 = 0.97, P < 0.001). Our results suggest that measurement of DNA adducts in WBCs may be an indicator of DNA adduct formation in BM following BZ exposure.

Oxidation of eugenol to form DNA adducts and 8-hydroxy-2'-deoxyguanosine: role of quinone methide derivative in DNA adduct formation.

We have investigated the activation of eugenol to form DNA adducts and oxidative base damage. Treatment of myeloperoxidase containing HL-60 cells with eugenol, produced a dose-dependent formation of three DNA adducts as detected with P1-enhanced 32P-post-labeling. Incubation of HL-60 cells with the combination of 100 microM eugenol and 100 microM H2O2 potentiated the levels of DNA adduct in HL-60 cells by 14-fold, which suggests peroxidase activation in adduct formation. In vitro activation of eugenol with either horseradish peroxidase or myeloperoxidase and H2O2 produced three DNA adducts that were inhibited by the addition of either ascorbic acid or glutathione, by 66 and 90%, respectively. The DNA adducts formed in HL-60 cells treated with eugenol were the same as those formed by in vitro peroxidase activation. In addition to adduct formation, peroxidase activation of eugenol produced a 2- to 3-fold increase in the level of oxidative base damage. Eugenol quinone methide was prepared by Ag(I)oxide oxidation of eugenol. Peroxidase activation of eugenol gave a product that had the same UV spectrum as eugenol quinone methide, which suggests that it was one of the products. Reaction of eugenol quinone methide with either DNA or deoxyguanosine-3'-phosphate produced two principal adducts (2 and 4). When DNA adduct 2 formed by incubation of eugenol quinone methide with deoxyguanosine-3'-phosphate was compared with DNA 2 adduct formed in HL-60 cells treated with eugenol results demonstrated that they were the same. This suggests that eugenol quinone methide is one of the reactive intermediates leading to DNA adduct formation in cells. Activation of eugenol with 10 microM copper sulfate resulted in the production of one principal (2) and several minor adducts. DNA adduct 2 formed by activation of eugenol with copper sulfate was the same as DNA adduct 2 formed by either peroxidase activation of eugenol or by reactions with eugenol quinone methide, which indicates that the reactive intermediates generated by these activation systems were similar. Copper sulfate produced a 95-fold increase in the level of oxidative base damage, which was significantly inhibited by the addition of either bathocuproinedisulphonic acid or catalase. The formation of oxidative base damage was consistent with a Fenton reaction mechanism. Our results demonstrate that eugenol can be activated to form both DNA adducts and oxidative base damage. We propose that the formation of this DNA damage may contribute to the observed toxic properties of eugenol.