James G. Omichinski

An automated alkaline elution system: DNA damage induced by 1,2-dibromo-3-chloropropane in vivo and in vitro

An automated alkaline elution system for the detection of DNA damage has been developed. After manual application of samples, which is completed within 5 min, the subsequent supply of liquids, changes in flow rates, and temperature are controlled automatically. The system operates 16 filters and may easily be expanded. The sensitivity of the fluorometric DNA determinations with the Hoechst 33258 dye is increased by using an elution buffer (20 mM Na2EDTA, pH 12.50) with low background fluorescence. DNA is determined using an automated setup similar to the one recently presented by Sterzel et al. (1985, Anal. Biochem. 147, 462-467). The most significant modification is the use of a neutralization buffer which allows variations in the pH of eluted fractions. This change increases the sensitivity of the DNA measurements. The automated alkaline elution system was evaluated using the nematocide 1,2-dibromo-3-chloropropane (DBCP) in a study of its genotoxic effects in the testes and the kidneys. Significant DNA damage was induced in testicular cells by 2.5 microM DBCP (1 h) in vitro and 85 mumol/kg DBCP ip (3 h) in vivo. The damage appeared after short treatment times (10 min in vivo). Variations in the observed DBCP response in vivo were largely due to interanimal variations. The automated alkaline elution system proved to be a sensitive assay also for the detection of DNA damage in kidney nuclei prepared from rats exposed to DBCP. Provided that kidney nuclei from untreated rats, mice, or hamster were kept ice-cold until lysing, 85-100% of their DNA was retained after 16 h of elution, indicating highly intact DNA. Under the same conditions, guinea pig DNA was rapidly degraded unless the nuclei were prepared in a buffer with a higher concentration of Na2EDTA (20 mM).

Protein kinase C-γ is present in adriamycin resistant HL-60 leukemia cells

The isoform pattern of protein kinase C (PKC) was examined in wild-type and Adriamycin-resistant (HL-60/AR) HL-60 leukemia cells. Analyses were carried out by immunoblotting with mouse monoclonal antibodies against PKC-alpha and PKC-beta and a rabbit polyclonal antibody against the variable (V3) region of PKC-gamma. HL-60/AR cells contained an equivalent level of PKC-alpha and a lower amount of PKC-beta than HL-60 cells. In contrast, only HL-60/AR cells contained PKC-gamma. These results indicate that the regulation of this family of isoenzymes is altered in drug-resistant cells.

Testicular necrosis and DNA damage caused by deuterated and methylated analogs of 1,2-dibromo-3-chloropropane in the rat

To study the role of metabolism in 1,2-dibromo-3-chloropropane (DBCP)-induced testicular damage in rats, selectively deuterated and methylated analogs of DBCP were given as a single ip dose of 340 mumol/kg and testicular toxicity was determined 10 days after treatment. None of the four deuterated analogs C1-D2-, C2-D1-, C3-D2-, or C1-C2-C3-D5-DBCP reduced the degree of testicular damage compared to DBCP, indicating that metabolic cleavage of a C-H bond was not rate-limiting in DBCP-induced testicular toxicity. Of the five methylated analogs, C1-methyl-, C1-dimethyl-, C2-methyl-, and C3-methyl-DBCP and 1,2-dibromo-4-chlorobutane, only C3-methyl-DBCP caused testicular toxicity. DBCP treatment resulted in increased testicular DNA damage at doses of 85-170 mumol/kg as measured by alkaline elution of DNA from testicular cells isolated 3 hr after in vivo treatment. The perdeutero-DBCP analog induced testicular DNA damage that was at least as extensive as that induced by DBCP. Of the methylated analogs tested, only C3-methyl-DBCP gave a marked dose-dependent increase in testicular DNA damage between 170 and 540 mumol/kg. There were no significant differences in the testicular tissue distribution between DBCP, perdeutero-DBCP, and the methylated DBCP analogs. Furthermore, in distribution studies with DBCP, C1-methyl- and C3-methyl-DBCP, and 1,2-dibromo-4-chlorobutane, the highest tissue concentrations were found in the kidneys, followed by the liver and then the testes. The fact that testicular DNA damage of DBCP and its deuterated and methylated analogs paralleled their ability to cause testicular necrosis and atrophy makes measurement of DNA damage a very useful correlate in mechanistic studies of DBCP-induced testicular cell death.

Species differences in testicular necrosis and DNA damage, distribution and metabolism of 1,2-dibromo-3-chloropropane (DBCP)

The human testicular toxicant 1,2-dibromo-3-chloropropane (DBCP) was studied for the same end-point in 4 different species of laboratory animals. Marked necrosis and atrophy of the seminiferous epithelium were observed in rats and guinea pigs 10 days after a single i.p. administration of DBCP (170-340 mumol/kg), whereas significantly less damage was observed in hamsters and mice. The testicular concentrations of DBCP measured at various time-points after the i.p. injection of DBCP indicated that factors in addition to tissue concentration were of importance for the observed species differences in sensitivity towards DBCP-induced testicular damage. Also, there did not seem to be any direct correlation between DBCP-induced in vivo testicular toxicity and in vitro GSH-dependent dehalogenation, inasmuch as the rate of bromide release from DBCP with hamster testicular cytosol was as fast as that with rat cytosol. Testicular DNA damage, as determined by alkaline elution 60 min after in vivo administration of 170 mumol/kg DBCP, was observed only in rats and guinea pigs. Thus, induction of DNA damage correlates with the relative susceptibilities of the species towards DBCP-induced testicular necrosis. To further study species differences in testicular activation of DBCP to DNA-damaging intermediate(s), cells isolated from the testes of the 4 species were incubated with DBCP. Testicular cells from rats and guinea pigs were the only preparations developing substantial DNA damage after 60 min incubation with low concentrations of DBCP (5-50 microM). The findings indicate that rats are sensitive towards DBCP-induced testicular necrosis because rat testicular cells easily activate DBCP to a DNA-damaging intermediate(s). The relative high testicular DBCP concentration as well as the ability to activate DBCP may explain the sensitivity of guinea pigs towards DBCP-induced testicular toxicity.

Mutagenic activity of halogenated propanes and propenes: effect of bromine and chlorine positioning

A series of halogenated propanes and propenes were studied for mutagenic effects in Salmonella typhimurium TA100 in the absence or presence of NADPH plus liver microsomes from phenobarbital-induced rats as an exogenous metabolism system. The cytotoxic and mutagenic effects of the halogenated propane 1,2-dibromo-3-chloropropane (DBCP) has previously been studied in our laboratories. These studies showed that metabolic activation of DBCP was required to exert its detrimental effects. All of the trihalogenated propane analogues were mutagenic when the microsomal activation system was included. The highest mutagenic activity was obtained with 1,2,3-tribromopropane, with approximately 50-fold higher activity than the least mutagenic trihalogenated propane, 1,2,3-trichloropropane. The order of mutagenicity was as follows: 1,2,3-tribromopropane > or = 1,2-dibromo- 3-chloropropane > 1,3-dibromo-2-chloropropane > or = 1,3-dichloro-2-bromopropane >> 1-bromo-2,3-dichloropropane > 1,2,3-trichloropropane. Compared to DBCP, the dihalogenated propanes were substantially less mutagenic. Only 1,2-dibromopropane was mutagenic and its mutagenic potential was approximately 1/30 of that of DBCP. In contrast to DBCP, 1,2-dibromopropane showed similar mutagenic activity with and without the addition of an activation system. The halogenated propenes 2,3-dibromopropene and 2-bromo-3-chloropropene were mutagenic to the bacteria both in the absence and presence of the activation system, whereas 2,3-dichloropropene did not show any mutagenic effect. The large differences in mutagenic potential between the various halogenated propanes and propenes are proposed to be due to the formation of different possible proximate and ultimate mutagenic metabolites resulting from the microsomal metabolism of the various halogenated propanes and propenes, and to differences in the rate of formation of the metabolites. Pathways are proposed for the formation of genotoxic metabolites of di- and trihalogenated propanes and dihalogenated propenes.

Detection and mechanism of formation of the potent direct-acting mutagen 2-bromoacrolein from 1,2-dibromo-3-chloropropane

The nematocide 1,2-dibromo-3-chloropropane (DBCP) was converted to products which are mutagenic for Salmonella typhimurium TA 100 in the presence of rat liver microsomes, NADPH, and oxygen. Typical in vivo and in vitro inhibitors of cytochrome P-450 decreased DBCP mutagenicity in the presence of microsomes. Addition of glutathione to cytosolic preparations failed to bioactivate DBCP to mutagenic metabolites. Mutagenicity studies with selectively deuterated analogs showed that substitution of deuterium for hydrogen at C-1 or C-3 of DBCP modestly decreased mutagenicity, but that deuteration at both C-1 and C-3 markedly decreased mutagenicity. The formation rates of the potent direct-acting mutagen, 2-bromoacrolein (2-BA), in incubations of DBCP and its deuterated analogs with rat liver microsomes, correlated with the isotope effects on mutagenicity. Characterization of 2-BA was accomplished by gas chromatography-mass spectrometry using positive-ion chemical ionization. Mass spectral analysis of 2-BA formed from specifically deuterated analogs of DBCP indicated that initial oxidative dehalogenation at C-1 followed by a spontaneous beta-elimination reaction was the preferred pathway in the formation of 2-BA from DBCP. These results demonstrate that mutagenic metabolites of DBCP are formed by cytochrome P-450-mediated oxidative metabolism, and that 2-BA is a major mutagen formed.

Renal necrosis and DNA damage caused by selectively deuterated and methylated analogs of 1,2-dibromo-3-chloropropane in the rat

Selectively deuterated and methylated analogs of the nematocide 1,2-dibromo-3-chloropropane (DBCP) were compared to DBCP in causing acute renal damage in rats. All of the six deuterated analogs tested at 340 mumol/kg, including the perdeutero compound, failed to significantly alter the kidney necrosis observed at 48 hr compared to DBCP. Furthermore, when the perdeutero analog was administered at several doses (42.5, 85, 170, and 340 mumol/kg), it caused kidney damage that was not significantly different than that caused by an equivalent molar dose of nondeuterated DBCP. Of the five methylated analogs tested at 170 and 340 mumol/kg, only C3-methyl-DBCP and 1,2-dibromo-4-chlorobutane caused nephrotoxicity. The C2-methyl-, C1-dimethyl-, and C2-methyl-DBCP analogs failed to cause renal necrosis determined 48 hr after dosing. In distribution studies DBCP, perdeutero-DBCP, and all the methylated analogs were found to concentrate in the kidney approximately 25 times relative to plasma 1 hr after administration. DBCP at doses of 4.3 mumol/kg and higher caused DNA damage in the kidney as early as 10 min after administration, as measured by alkaline elution of DNA from isolated kidney nuclear preparations. Perdeuteration did not decrease the DNA damaging effect of DBCP. The ability of the methylated DBCP analogs to induce renal DNA damage correlated with their necrogenic potential. Experiments using pretreatments that are known to decrease the nephrotoxicity caused by glutathione and cysteine conjugates of several halogenated alkenes were conducted to examine the effect of these pretreatments on DBCP-induced nephrotoxicity. Probenecid, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) and aminooxyacetic acid did not significantly alter renal necrosis or DNA damage induced by DBCP. Based on the absence of any significant isotope effects with the predeutero-DBCP analog, it appears that breaking of a carbon-hydrogen bond is not the rate-limiting step in DBCP-induced nephrotoxicity. Studies with the methylated DBCP analogs indicate that a vicinal dibromo ethyl group must minimally be present for nephrotoxic potential. Furthermore, it seems unlikely that metabolism by renal cysteine conjugate beta-lyase is rate-limiting for DBCP nephrotoxicity.

Metabolism in vitro of tris(2,3-dibromopropyl)- phosphate: Oxidative debromination and bis(2,3- dibromopropyl)phosphate formation as correlates of mutagenicity and covalent protein binding

Tris(2,3-dibromopropyl)phosphate (Tris-BP) was found to be metabolized by liver microsomes obtained from untreated and phenobarbital-pretreated rats. Metabolites of Tris-BP, whose formation was dependent on NADPH and oxygen, included bromide ion and bis(2,3-dibromopropyl)phosphate (Bis-BP). The rates of formation of these metabolites were markedly increased in liver microsomes isolated from phenobarbital-pretreated rats compared to microsomes from untreated rats. In the presence of either SKF 525-A or metyrapone, the formation rates of bromide ion and Bis-BP were decreased, whereas alpha-naphthoflavone had no effect. The effects of the various treatments on bromide release and Bis-BP formation paralleled those that have been previously observed with respect to the activation of Tris-BP to mutagenic and covalently protein bound metabolites. Furthermore, rates of oxidative debromination of several Tris-BP analogs directly correlated with their respective mutagenicities. Addition of glutathione (GSH) to microsomal incubations of Tris-BP increased bromide release substantially over control, values but had no effect on Bis-BP formation. On the other hand, the addition of GSH to microsomes decreased covalent binding and mutagenicity of Tris-BP with increased formation of water soluble metabolites. GC/MS analysis of ethyl acetate extracts from incubations of rat liver microsomes with Tris-BP identified 2-bromoacrolein (2-BA) as a metabolite. Introducing deuterium at the carbon atom number 1 of the propyl moiety of Tris-BP had no effect on either bromide release or mutagenicity, whereas the analog labelled at carbon atom 3 showed significant isotope effects on both activities. In contrast, deuterium substitution at carbon atom 2 gave a significant isotope effect on bromide release, but not on mutagenicity. The data indicate that Tris-BP can be metabolized by rat liver microsomes to Bis-BP and 2-bromoacrolein catalyzed by cytochrome P-450 in a process liberating bromide ions. Further, the results are consistent with oxidation at the terminal carbon atom of Tris-BP thereby forming 2-bromoacrolein, which is postulated to be the metabolite mainly responsible for Tris-BP mutagenicity.

Role of P-450 activity and glutathione levels in 1,2-dibromo-3-chloropropane tissue distribution, renal necrosis and in vivo DNA damage

Treatments known to alter P-450 activity and glutathione levels were used to elucidate the involvement of P-450 and glutathione S-transferase metabolism in 1,2-dibromo-3-chloropropane (DBCP) organ toxicity in the rat. Phenobarbital pretreatment abolished DBCP-induced renal necrosis, whereas it had only a small effect on initial renal DNA damage. The DBCP levels in plasma and tissues were markedly reduced by phenobarbital pretreatment. Perdeuterated DBCP had much higher plasma and tissue levels than protio-DBCP in phenobarbital-pretreated animals, but perdeuteration was without effect in uninduced animals. This indicates that P-450 metabolism of DBCP is of major importance only in phenobarbital-pretreated animals. In order to study the effects of decreased glutathione levels on renal distribution and toxicity, rats were pretreated with either diethyl maleate or buthionine sulfoximine. The DBCP levels in plasma and tissues showed transitory elevations after diethyl maleate and buthionine sulfoximine pretreatment compared to the control situation. Despite the fact that diethyl maleate and buthionine sulfoximine pretreatments are known to block DBCP-induced DNA damage in vitro, these pretreatments did not significantly alter DBCP-induced renal necrosis nor DNA damage. Thus, a role for glutathione conjugation in DBCP-induced in vivo renal toxicity could not be established in the present study.

Activation mechanism of Tris(2,3-dibromopropyl)phosphate to the potent mutagen, 2-bromoacrolein

The potent mutagen 2- bromoacrolein is formed from the carcinogenic flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) on incubation with hepatic microsomes. Substitution of deuterium for hydrogen at the terminal carbon atoms (C-3) of Tris-BP significantly decreased both the mutagenic response and the formation rate of 2- bromoacrolein . Mass spectral analysis of the 2- bromoacrolein that was formed from the selectively deuterated analogs of Tris-BP revealed that the primary mechanism for the formation of 2- bromoacrolein involves an initial oxidative dehalogenation at C-3 followed by a beta-elimination reaction.