Recognizing toxicokinetic and toxicodynamic variability is important in risk assessment of chemicals and may help to explain interindividual differences in susceptibility in exposed populations. Both toxicokinetic and toxicodynamic factors may be influenced by age and disease processes and show genetic polymorphic variation. Decreased metabolic activity in the very young or very old may enhance chemical toxicity caused by the parent chemical. Similarly, disease processes affecting hepatic metabolism and renal excretion may delay inactivation of many xenobiotics. Genetic polymorphisms may enhance toxicity in rapid metabolizers when the toxicity is caused by a reactive intermediate and increase toxicity in slow metabolizers when the toxicity is caused by a parent chemical. Some cells of the developing conceptus are exquisitely sensitive to chemical exposure. Also, organs and tissues of newborns and elderly individuals may show increased responses toward xenobiotics. In addition, disease-induced altered receptor sensitivity and tissue repair may result in enhanced chemical toxicity. Further, tissue antioxidant defense against radical damage may be compromised under nutritional deficiencies and starvation. Hereditary peculiarities in individual responses to environmental chemicals may be due to polymorphic variation of receptor proteins and tissue repair enzymes, although the database for such variation is quite limited.>

Several recent studies indicate declines in sperm production, as well as increases in the incidence of genitourinary abnormalities such as testicular cancer, cryptorchidism and hypospadias (Toppari et al., 1996). It is not known if these effects are due to exposure to chemical pollutants or if other ethiological factors are involved. Animal studies indicate that chemicals will induce such effects by various genetic, epigenetic or non-genetic mechanisms. Recently, much attention has been focused on embryonic/fetal exposure to oestrogen-mimicking chemicals (Toppari et al., 1996). However, the possibility that chemicals may cause reproductive toxicity by other mechanisms such as interactions with DNA, should not be ignored. DNA damage in germ cells may lead to the production of mutated spermatozoa, which in turn may result in spontaneous abortions, malformations and/or genetic defects in the offspring. Regarding the consequences of DNA alterations for carcinogenesis it is possible that genetic damage may occur germ cells, but the consequences are not expressed until certain genetic events occur in postnatal life. Transmission of genetic risk is best demonstrated by cancer-prone disorders such as hereditary retinoblastoma and the Li-Fraumeni syndrome. A number of experiments indicate that germ cells and proliferating cells may be particularly sensitive to DNA damaging agents compared to other cells (Masters et al., 1993; Holme et al., 1997).

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK1, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30–300 µmol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK1 cells (100 µmol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 µmol/L. Flow cytometric analysis showed that DBCP (1–10 µmol/L) exposure for 24 h caused an accumulation of LLCPK1 cells in the G2/M-phase. In HL-60 cells the accumulation in G2/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK1 cells exposed to 100–500 µmol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G1 cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK1 cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G2/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.

Preparations of testicular cells from human organ transplant donors and from Wistar rats were compared with respect to their composition of the different testicular cell types, their ability to metabolize 1,2-dibromo-3-chloropropane (DBCP), and their relative sensitivity to induction of DNA single strand breaks and alkali labile sites (ssDNA breaks) after treatment with DBCP, 4-nitroquinoline N-oxide (4-NQO), and x rays. Flow cytometric and microscopic analysis demonstrated that the interindividual variation in the composition of testicular cell types was considerably greater in the human tissue than in that from rats. The in vitro metabolic activation of DBCP (50 to 250 μM), measured as radiolabel covalently bound to macromolecules, was three-fold faster in rat testicular cells compared to human testicular cells. X rays (1 to 10 Gy) and 4-NQO (0.5 to 2.5 μM) induced ssDNA breaks to a similar extent in both human and rat testicular cells as measured by single cell gel electrophoresis (SCGE) and alkaline niter elution. In contrast, 1,2-dibromo-3-chloropropane (DBCP) (3 to 300 μM) caused no significant DNA damage in human testicular cells, whereas in rats there was a clear concentration-dependent increase in ssDNA breaks. The data show that, compared to rats, testicular cells from humans are less efficient in activating DBCP to metabolites binding covalently to macromolecules. However, from the rate of covalent binding observed one would expect a significant degree of DBCP-induced ssDNA breaks in the human testicular cells. The low level of DBCP-induced ssDNA breaks in human testicular cells could indicate that different reactive DBCP metabolites are involved in binding to cellular macromolecules compared to DNA damage, or that different rates of DNA repair exist in human and rat testicular cells.

An in vivo genotoxicity assay system based on alkaline elution has been used to study the formation and removal of DNA damage induced by 1,2-dibromo-3-chloropropane (DBCP). Cells/nuclei from different tissues and organs of Wistar rats were prepared by a rapid mincing/homogenization technique. Thirty-six samples of which up to 11 were from different organs of the same animal, were then assayed in parallel for DNA damage (DNA singlestrand breaks plus alkali-labile sites = SSBs) with a semi-automated alkaline elulion system. A single i.p. injection of DBCP gave dose- (5 and 10 mg/kg) and time- (20 min–4 h) dependent SSBs in kidney and liver DNA from male rats. At 10 mg/kg DBCP, SSBs were formed in all organs examined except the bone marrow and colon; however, an increased dose of 40 mg/kg produced SSBs also in the latter two organs. The relative susceptibilities to DBCP-induced DNA damage were: kidney ~ duodenum > liver > lung ~ brain ~ urinary bladder ~ glandular stomach > spleen ~ testis > bone marrow ~ colon. These relative levels correlate with previous data on tissue distribution and organ necrosis in liver, kidney and testis of rats given a single i.p. dose of DBCP. When female rats were injected i.p. with 5, 10 or 20 mg/kg (nonhepatotoxic doses) at day 20 of pregnancy, similar levels of SSBs were detected in the livers of the dam and the fetuses. In adult male rats, time-dependent changes in SSBs were followed in the liver and kidney after DBCP exposure. In both organs SSBs peaked around 4 h postexposure, 50% had been removed by 12–24 h, whereas at day 2–3 SSB frequencies had returned to control levels. Pretreatment of rats with phenobarbital prior to DBCP exposure reduced the maximum level of DNA damage as well as its persistence. In cultured primary hepatocytes from male rats exposed in vitro to DBCP (2–20 μM, 1 h), 50% of the initial DNA damage had been repaired within ~ 100 min. In conclusion, the experiments indicate that the distribution characteristics of DBCP are of major importance for DNA damage and its persistence in various organs of rats. The data are also in accordance with glutathione-S-transferase, rather than P450, being the most important pathway for metabolic activation of DBCP in rat extrahepatic tissues including the fetal liver. It appears that alkaline elution of cells/nuclei prepared from exposed animals constitutes a sensitive, rapid and versatile technique to study organ- and cell-specific genotoxicity in vivo.

Testicular cells prepared from human organ transplant donors or from Wistar rats were used to compare 15 known reproductive toxicants with respect to their ability to induce DNA damage, measured as single-strand DNA breaks and alkali labile sites (ssDNA breaks) with alkaline filter elution. The compounds tested included various categories of chemicals (i.e., pesticides, industrial chemicals, cytostatics, and mycotoxins) most of which are directly acting genotoxicants (i.e., reacting with DNA either spontaneously or via metabolic activation). In addition, a few indirect genotoxic and nongenotoxic reproductive toxicants were included. Six of the chemicals induced no significant levels of ssDNA breaks in human and rat testicular cells: methoxychlor (10 to 100 μM, human and rat), benomyl (10 to 100 μM, human and rat), thiotepa (10 to 1000 μM, human and rat), cisplatin (30 to 1000 μM, human; 100 to 1000 μM, rat), Cd2+ (30 to 1000 μM, human; 100 to 1000 μM, rat), and acrylonitrile (30 to 1000 μM, human; 30 to 300 μM, rat). Four chemicals induced significant levels of ssDNA breaks in testicular cells from both species: styrene oxide (≥ 100 μM, rat and human), 1,2-dibromoethane (EDB) (≥ 100 μM, rat; 1000 μM human), thiram (≥ 30 μM, rat; ≥ 100 μM, human), and chlordecone (300 μM, rat; ≥ 300 μM, human). Finally, five chemicals induced ssDNA breaks in one of the two species. Four chemicals induced significant ssDNA breaks in rat testicular cells only: 1,2-dibromo-3-chloropropane (DBCP) (≥ 10 μM), 1,3-dinitrobenzene (1,3-DNB) (≥ 300 μM), Cr6+ (1000 μM), and aflatoxin B1 (≥ 100 μM), the last two of these produced only a minor positive response. One chemical, acrylamide, induced a marginal increase in ssDNA breaks in human at 1000 μM, but not in rat testicular cells. Although based on a limited number of donors, the data indicate a close correlation between the induction of DNA damage in human and rat testicular cells in vitro. For some chemicals, however, there appears to be differences in the susceptibility to chemically induced ssDNA breaks of isolated testicular cells from the two species. The data indicate that the parallel use of human and rat testicular cells provides a valuable tool in the assessment of human testicular toxicity.

The metabolism of 1,2-dibromo-3-chloropropane (DBCP), measured as the formation of water soluble metabolites and metabolites covalently bound to macromolecules, was studied in isolated rat liver, kidney, and testicular cells, in subcellular fractions, and with purified rat and human glutathione S-transferases (GSTs). The rate of formation of water soluble metabolites in the cells were in the order liver > kidney > testis. The rate of covalent macromolecular binding of reactive DBCP metabolites in the different cell types was of the same relative order. Pretreatment of the cells with the GSH depleting agent diethyl maleate (DEM) markedly decreased the rate of covalent binding in all cell types. Both the overall metabolism and the formation of DBCP metabolites that covalently bound to macromolecules, were substantially higher in rat testicular cells compared to hamster testicular cells. Rat liver cytosol and microsomes, and various purified rat and human GSTs extensively metabolized DBCP to water soluble metabolites in the presence of GSH. When compared to isolated cells, substantially lower rates of binding per mg protein could be observed in subcellular fractions. Binding of DBCP was detected in the microsomal and cytosolic fractions in the absence of NADPH, though in microsomes fortified with a NADPH-regenerating system, the generation of reactive DBCP metabolites was approximately doubled. Studies with purified rat GST isozymes showed that the relative overall GSH conjugation activity with DBCP was in the following order: GST form 3-3 > 2-2 approximately 12-12 > 1-1 > 4-4 approximately 8-8 approximately 7-7. Furthermore, human GST forms also readily metabolized DBCP with activities of GST A1-2 > A2-2 approximately A1-1 > M1a-1a > M3-3 approximately P1-1.

1,2-Dibromo-3-chloropropane (DBCP)-induced toxicity was studied in rat germ cells from different stages of spermatogenesis, separated by centrifugal elutriation, and in Sertoli cells prepared from sexually mature and immature animals. The in vitro metabolic activation of 50 to 250 μM DBCP, measured as covalent binding of 14C-DBCP to macromolecules, was highest in round spermatids, lowest in Sertoli cells and elongating/ elongated spermatids, and intermediate in spermatocytes. High concentrations of DBCP (≥ 250 μM) caused a decrease in oxygen consumption and mitochondrial rhodamine 123 uptake, indicating an effect on mitochondrial function. Altered Sertoli cell function, measured as detachment of germ cells in Sertoli-germ cell cocultures, was evident at DBCP concentrations ≥ 300 μM. DBCP-induced DNA damage occurred at much lower concentrations (10 to 30 μM) when compared to effects on mitochondrial function and Sertoli cell function. The extent of single strand DNA breaks and alkali-labile sites (ssDNA breaks) measured by the alkaline filter elution technique and the single cell gel electrophoresis assay, were greatest in the round spermatids >spermatocytes = Sertoli cells>elongating/elongated spermatids. The study demonstrates that various testicular cell types show differences in their rates of activation of DBCP to metabolites that bind to macromolecules. DNA is a more sensitive intracellular target in DBCP-induced testicular toxicity than mitochondria. Round spermatids appear to be more susceptible to DBCP-induced ssDNA breaks than spermatocytes, elongating/ elongated spermatids, or Sertoli cells.

The genotoxicity of paracetamol, including covalent binding to DNA, induction of DNA single-strand breaks (SSBs), and inhibition of replicative and repair synthesis of DNA, has been investigated in rodents in vivo. In the covalent binding studies male ICR mice were fasted and pretreated with diethyl maleate to deplete hepatic glutathione (GSH) and 300 mg/kg of [G-3H]paracetamol was administered intraperitoneally (i.p.). Animals were killed at 2, 6, 24, 72 and 168 h after paracetamol and hepatic or renal DNA and protein were isolated and the extent of covalent binding determined. Maximal binding to liver DNA, 8.4 +/- 3.1 pmol/mg of DNA, was observed at 2 h and declined rapidly to 2.6 pmol/mg at 24 h. Measurable binding (1.4 pmol/mg of DNA) was detected at 7 days. Protein binding in the liver in these animals peaked between 2 and 6 h (887 pmol/mg of protein at 2 h) and declined monoexponentially to 52 pmol/mg at 7 days. Although based on a limited body of data, covalent binding was also detected in DNA isolated from the kidney. DNA damage measured as SSBs by alkaline elution was induced in nuclear DNA isolated from the liver but not from the kidney, 2 h after i.p. injection of paracetamol at 600 mg/kg in male B6 mice. Only marginal DNA damage was noted at 300 mg/kg. The alkaline elution profile from damaged liver nuclei was markedly biphasic, suggesting that breaks were induced in DNA from a subpopulation of liver cells. The non-hepatotoxic paracetamol regioisomer, acetyl-m-aminophenol (600 mg/kg), which binds covalently to proteins, did not cause DNA SSBs.(ABSTRACT TRUNCATED AT 250 WORDS)

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 ≥ 1,2-dibromo-3-chloropropane > 1,3-dibromo-2-chloropropane ≥ 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 130 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.

Analogs of tris(2,3-dibromopropyl)phosphate (Tris-BP) either labeled at specific positions with carbon-14, phosphorus-32, or oxygen-18 or dual-labeled with both deuterium and tritium were used as metabolic probes to study the chemical and metabolic events in the bioactivation of Tris-BP to chemically reactive metabolites in liver microsomal preparations. Oxidation at the terminal (C-3) carbon atom of the propyl groups of Tris-BP yielded the direct-acting mutagen 2-bromoacrolein as the major metabolite that binds to DNA. Although this reactive metabolite also appears to bind to microsomal protein, the rate of binding of radiolabeled Tris-BP to protein is 15-20x greater than binding to DNA, and some metabolites that retain the phosphate group are bound. Studies with deuterated analogs of Tris-BP implicate oxidation at C-2 of the propyl group as a major pathway that leads to protein binding which is enhanced by phenobarbital pretreatment of rats. Moreover, investigations with 18O-Tris-BP and H2(18)O show that Bis-BP that is formed from oxidation of Tris-BP incorporates one atom of oxygen from water. Deuterium isotope studies suggest that most of the Bis-BP arises from initial oxidation at C-2. Taken together these studies indicate that P-450 oxidation of Tris-BP at C-2 of the propyl group yields a reactive alpha-bromoketone metabolite of Tris-BP that can either alkylate proteins directly or be hydrolyzed to Bis-BP and an alpha-bromo-alpha'-hydroxyketone that can alkylate microsomal proteins.

By use of an isolation procedure including centrifugal elutriation and density gradient centrifugation, relatively pure fractions of Clara cells and type II cells were obtained from rabbit lungs. These cells and alveolar macrophages isolated by lavage were exposed to methyl methanesulfonate (MMS), 1,2-dibromo-3-chloropropane (DBCP), 1-nitropyrene (1-NP), 2-nitrofluorene (2-NF), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosonornicotine (NNN), N-nitrosoheptamethyleneimine (NHMI) or phorbol 12-myristate 13-acetate (TPA). DNA damage measured as alkali-labile sites and/or single-strand breaks was then determined in the different lung cells by an automated alkaline elution system. The direct-acting compound MMS showed similar DNA-damaging effect in Clara cells, type II cells and alveolar macrophages. The nematocide DBCP, activated by both P450- and glutathione S-transferase(s)-dependent pathways, caused considerably less DNA damage in macrophages than in Clara or type II cells. Similar differences between the lung cells in induction of DNA damage as observed with DBCP were demonstrated after exposure to the activation-dependent nitrosamines NNK and NHMI and the tumor promoter TPA. The other test substances (1-NP, 2-NF, NNN) did not caused by marked DNA damage measured by the alkaline elution technique. These findings are in agreement with the known metabolic capacity of these cell types, indicating that Clara and type II cells are possible primary targets for lung toxic/carcinogenic compounds.

Analogs of tris(2,3-dibromopropyl)phosphate (Tris-BP) either labeled at specific positions with carbon-14 and phosphorus-32 or dual-labeled with both deuterium and tritium were administered to male Wistar rats at a nephrotoxic dose of 360 μmol/kg. The covalent binding of Tris-BP metabolites to hepatic, renal, and testicular proteins was determined after 9 and 24 hr, and plasma concentrations of bis(2,3-dibromopropyl)-phosphate (Bis-BP) formed metabolically from Tris-BP were measured at intervals throughout the initial 9-hr postdosing period. The covalent binding of 14C-Tris-BP metabolites in the kidney (2495 ± 404 pmol/mg protein) was greater than that in the liver (476 ± 123 pmol/mg protein) or testes (94 ± 11 pmol/mg protein); the extent of renal covalent protein binding of TrisBP metabolites was decreased by 82 and 84% when deuterium was substituted at carbon-2 and carbon-3, respectively. Substitution of Tris-BP with deuterium at carbon-2 or carbon-3 also decreased the mean area under the curve for Bis-BP plasma concentration by 48 and 57%, respectively. The mechanism of TrisBP-induced renal and hepatic DNA damage was evaluated in Wistar rats by an automated alkaline elution procedure after the administration of analogs of Tris-BP or Bis-BP labeled at specific positions with deuterium. Renal DNA damage was decreased when Tris-BP was substituted with deuterium at either carbon-2 or carbon-3; the magnitude of the change correlated with both a decrease in the area under the Bis-BP plasma curve and a decrease in renal covalent binding of Tris-BP metabolites for each of the deuterated analogs. In marked contrast, analogs of Bis-BP labeled with deuterium at carbon-2 or carbon-3 did not show a decrease in the severity of renal DNA damage compared to unlabeled Bis-BP. On the basis of these observations a metabolic scheme for hepatic P-450-mediated oxidation at either carbon-2 or carbon-3 of Tris-BP affording Bis-BP by two alternate pathways that are susceptible to primary deuterium kinetic isotope effects is proposed. The Tris-BP metabolite, BisBP, is subsequently metabolized to reactive intermediates that cause DNA damage and bind to kidney proteins in a mechanism independent of cytochrome P-450.

We have expressed human glutathione S-transferases GSTA1-1 and GSTP1-1 in Salmonella typhimurium TA100 in order to assess the ability of these enzymes to modulate the mutagenicity of 1,2-dibromo-3-chloropropane (DBCP) and tris(2,3-dibromopropyl)phosphate (Tris-BP). Both compounds were mutagenic when activated by Aroclor-induced rat liver microsomes. However, when Aroclor-induced rat liver microsomes were used together with the GST-expressing strains the mutagenicity of both DBCP and Tris-BP was markedly potentiated. Neither of the GST-expressing strains potentiated the mutagenicity in the absence of microsomes, indicating that cytochrome P450-mediated metabolism was a prerequisite for GST-mediated potentiation. With DBCP both isozymes had comparable effects on mutagenic frequency, although the highest dose of DBCP was toxic in strains expressing GSTP1-1. In the case of Tris-BP, GSTP1-1 was much more active in potentiating the mutagenicity. These results indicate that human GSTs can play an important role in the activation of compounds such as DBCP and Tris-BP to mutagenic metabolites.

Benz[j]aceanthrylene (B[j]A) and benz[l]aceanthrylene (B[l]A), two isomeric cyclopenta polycyclic aromatic hydrocarbons (CP-PAH) structurally related to 3-methylcholanthrene, were studied with respect to their genotoxic effects in isolated liver and lung cells. Both compounds were found to cause DNA adducts measured by the 32P-postlabelling technique. The level of DNA-adducts in rat hepatocytes exposed to 30 micrograms/ml B[l]A and B[j]A for 4 h were 46.5 +/- 22.0 and 8.3 +/- 5.1 fmol/micrograms DNA respectively. Using butanol extractions, the major and one of the minor B[j]A adducts co-chromatographed with B[j]A-1,2-oxide adducts of 2'-deoxyadenosine and 2'-deoxyguanosine. Thus, oxidation at the cyclopenta-ring of B[j]A appears to be an important activation pathway. In hepatocytes, 3-30 micrograms/ml of B[j]A and B[l]A induced DNA damage and repair measured both as increased alkaline elution of DNA and as increased incorporation of [3H]TdR in the DNA. B[l]A was somewhat more potent than B[j]A in inducing DNA repair. Reactive CP-PAH intermediates formed in the hepatocytes caused mutations in Salmonella typhimurium TA98 upon co-incubation. DNA adducts were also observed in isolated rabbit lung cells exposed to 30 micrograms/ml B[l]A or B[j]A for 2 h. A total of 14.5 +/- 6.9, 2.9 +/- 2.1 and 0.2 +/- 0.6 fmol B[l]A adducts/micrograms DNA were observed in Clara cells, type II pneumocytes and alveolar macrophages respectively. The main B[l]A adduct observed in the liver cells was not found in the lung cells. On the other hand, the levels of B[j]A adducts in the lung cells were in the range 4-14% of that found in liver cells, and no major differences between the various lung cells were observed. Neither B[l]A nor B[j]A induced DNA damage measured by alkaline elution in the lung cells, indicating that these adducts are not alkali labile.

The organ specificity of tris(2,3-dibromopropyl)phosphate(Tris-BP)-induced DNA damage was investigated in the rat 2 h after a single i.p. injection of 350 mumol/kg. Extensive DNA damage, measured with the alkaline elution method, was found in the kidney, liver and small intestine. Less, but significant DNA damage was detected in the brain, lung, spleen, large intestine and testis. The role of different pathways in the activation of Tris-BP to DNA damaging products was studied in isolated liver and testicular cells. Concentrations as low as 2.5-5 microM Tris-BP caused DNA damage in the hepatocytes, whereas an approximately 10-fold higher concentration was needed in testicular cells to produce a similar amount of DNA damage. Depletion of GSH by diethyl maleate (DEM) did not affect the extent of DNA damage caused by Tris-BP in the liver cells, but blocked the genotoxic effect in testicular cells. Two specifically deuterated Tris-BP analogs, C3D2-Tris-BP and C2D1-Tris-BP, were significantly less potent in causing DNA damage than the protio compound in isolated liver cells and were somewhat less potent in testicular cells. The major urinary metabolite of Tris-BP, bis(2,3-dibromopropyl)phosphate (Bis-BP), was less potent than Tris-BP in causing kidney DNA damage after in vivo exposure. Furthermore, Bis-BP induced substantially less DNA damage in isolated liver and testicular cells. Similar to the effect of DEM on the DNA damage caused by Tris-BP, the DNA damage caused by Bis-BP could be decreased by DEM-pretreatment in testicular cells but not in liver cells. The present study shows that Tris-BP is a potent multiorgan genotoxic agent in vivo. The in vitro data indicate that P-450 mediated metabolism of Tris-BP is more important than activation by glutathione S-transferases of Tris-BP in liver cells, whereas the latter activation pathway seems to be most important in testicular cells.

The many different cell types identified in the lungs show a broad variation in morphology, function and metabolic capacity. Because of this variation certain cell types are more susceptible to the toxic insult of chemical exposures. The bronchiolar Clara cells and the alveolar type II cells are known to express relatively high levels of the P450 system compared to other pulmonary cell types (Boyd 1977; Philpot and Smith 1984; Minchin 1983). These cell types also appear to be target cells for several chemicals activated by P450 dependent reactions (Boyd and Reznik-SchüTler 1984). Furthermore, Clara and type II cells are able to proliferate. Of particular interest for activation of a number of known lung carcinogens seems to be the expression of the P450 IIB1 gene in Clara and type II cells making them possible primary target cells for initiation of lung carcinogenesis (Belinsky et al. 1989; Becher et al. 1991).