Lack of mammalian mutagenicity of the potent bacterial mutagen tris(2,3-dibromopropyl) phosphate and its metabolite 2-bromoacrolein.

The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and its metabolite 2-bromoacrolein (2BA) are very potent bacterial mutagens in Salmonella typhimurium (S. typhimurium) TA 100. In this study, we showed that 2BA and Tris-BP are also mutagenic in S. typhimurium TA 104, which detects mutations at AT base pairs, while TA 100 detects mutations at CG basepairs. We also studied the mutagenicity of 2BA in mammalian cells in vitro and in the rat in vivo. Firstly, 2BA was tested in the human lymphoblastoid cell line TK6. The results showed that there was no increase in mutation frequency at the hprt locus, whereas there was a large decrease in cell survival. Secondly, a shuttle vector system was used to study the induction of mutations by 2BA:DNA adducts. The vector was modified by insertion of a single-stranded oligonucleotide containing on average one 2BA:DNA adduct. No increase in mutation frequency above background was detected after replication of this vector in SV40 transformed normal human fibroblasts. Because the liver is a major site for bioactivation of Tris-BP to 2BA in vivo, we tested the initiating capacity of Tris-BP in the rat liver in a modified Solt & Farber initiation and promotion system. Administration of Tris-BP resulted in a small increase in the number of preneoplastic gamma-glutamyl-transpeptidase positive (GGT+) foci in the liver compared to control animals (only significant in the lowest size class). Modification of the experimental protocol by performing partial hepatectomy 24 h after the administration of Tris-BP, did not increase the number of GGT+ or glutathione S-transferase-P (GST-P+) positive foci above the control level. Taken together, these results indicate that, in spite of a high mutagenicity in S. typhimurium, 2BA and Tris-BP have low or negligible mutagenic effects in mammalian systems. The lack of mutagenic activity may explain why Tris-BP is not a carcinogen in the rat liver.

Deuterium isotope effect on the metabolism of the flame retardant tris(2,3-dibromopropyl) phosphate in the isolated perfused rat liver.

The metabolism of tris(2,3-dibromopropyl) phosphate (Tris-BP) was compared with that of completely deuterated Tris-BP (D15-Tris-BP) in an isolated, recirculating rat liver perfusion system in order to determine the relative quantitative importance of two different biotransformation pathways of Tris-BP: (i) cytochrome P450-mediated metabolism and (ii) GSH S-transferase-mediated metabolism. To accomplish this we quantitated the biliary excretion of S-(3-hydroxypropyl)glutathione (GSOH) as a marker metabolite for cytochrome P450-mediated metabolism and that of S-(2,3-dihydroxypropyl) glutathione (GSOHOH) as a marker metabolite for GSH S-transferase-mediated metabolism. Complete deuterium substitution of Tris-BP significantly decreased the formation of GSOH, whereas there was no effect on the formation of GSOHOH. Because our previous studies showed a large decrease in genotoxicity of D15-Tris-BP compared to Tris-BP, the present results support our hypothesis that cytochrome P450-mediated metabolism is responsible for the genotoxic effects of Tris-BP in the rat liver.

Metabolism and genotoxicity of the halogenated alkyl compound tris(2,3-dibromopropyl)phosphate.

1. The genotoxicity of the flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) was studied in vivo. Results showed that Tris-BP was highly clastogenic, but it could only initiate a low number of preneoplastic foci in the rat liver in vivo. In Drosophila, Tris-BP could be classified as a cross-linking agent, because it was more clastogenic than mutagenic. The use of completely deuterated Tris-BP as a metabolic probe revealed that cytochrome P450 and most likely the formation of 2-bromoacrolein (2BA) from Tris-BP is important for the observed genotoxic effects. 2. In contrast to the high mutagenicity of Tris-BP and 2BA in Salmonella typhimurium, we were unable to detect an increase in mutation frequency of 2BA on the hprt locus of human TK6 cell line. In another system, using a shuttle vector modified with 2BA:DNA-adducts, also no increase in mutation frequency could be detected in human cells. This low mutagenicity of 2BA corresponds with its low mutagenicity in Drosophila and its low induction of preneoplastic foci in the rat liver. 3. Several DNA adducts of 2BA have been identified, including an unstable 3-(bromooxypropyl)thymidine adduct which has the potential to form cross-links and a cyclic 3,N4-(bromo)propeno-deoxycytidine adduct which can possibly be involved in the clastogenicity of Tris-BP. 4. Taken together, these data indicate that Tris-BP and 2BA may not effectively induce gene mutations in eukaryotic systems, but rather be potent clastogens. Risk assessment of these and related compounds should therefore be based on the knowledge of clastogens rather than mutagens.

Genotoxicity of the flame retardant tris(2,3-dibromopropyl)phosphate in the rat and Drosophila: effects of deuterium substitution.

Formation of 2-bromoacrolein (2BA) from tris(2,3-dibromopropyl)phosphate (Tris-BP) by cytochrome P450 (cyt. P450) activity is most likely responsible for the high mutagenicity of Tris-BP for bacteria in vitro when rat liver microsomes are used for metabolic activation. As yet, it has not been established whether cyt. P450 plays a role in the formation of genotoxic metabolites from Tris-BP in higher organisms in vivo. This was studied by comparing the effects of completely deuterated Tris-BP (D15-Tris-BP) with normal Tris-BP in various in vivo test systems for genotoxicity: deuterium substitution should decrease cyt. P450-dependent bioactivation of Tris-BP. Three test systems in Drosophila were used to measure different types of genetic damage: (I) the ring-X chromosome loss (CL) test to detect clastogenicity, (II) the sex-linked recessive lethal (RL) test to detect forward mutations and deletions and (III) the white/white+ (w/w+) eye mosaic assay to detect predominantly mitotic recombination. Tris-BP was positive in all test systems, while D15-Tris-BP was without effect. The relative clastogenic efficiency of a compound, defined as the ratio of CL over RL, can be used to distinguish monofunctional agents (CL/RL ratio < or = 1) from those having cross-linking potential (CL/RL ratio > 2). The CL/RL ratio of Tris-BP was 2.4, indicating that Tris-BP has cross-linking potential. No CL/RL ratio for D15-Tris-BP could be determined, because it was negative in both tests. The putative Tris-BP metabolite 2BA was also tested in the Drosophila test systems. 2BA was positive in the CL test at a high dose, but it showed no response in the RL test. The relative clastogenic efficiency (the ratio of CL/RL) could not be determined accurately, but the data nevertheless argue in favour of cross-linking properties of 2BA. Further, 2BA was clearly positive in the w/w+ eye mosaic test system, which indicates that it is a recombinagen. Genotoxicity of Tris-BP in rats was determined by the induction of micronuclei in hepatocytes. Tris-BP administered intraperitoneally 17 h after 2/3 partial hepatectomy (PH), induced a high frequency of micronuclei at days 2 and 3 after injection (26% and 22%, respectively). D15-Tris-BP, however did not increase the micronuclei frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

Blocking of in vitro DNA replication by deoxycytidine adducts of the mutagen and clastogen 2-bromoacrolein.

Calf thymus single-stranded DNA was modified with 2-bromoacrolein (2BA), a genotoxic metabolite of tris(2,3-dibromopropyl)phosphate. This DNA was used as a template for in vitro DNA replication by T7-polymerase and Klenow fragment of Escherichia coli DNA polymerase I. Increasing levels of 2BA modification led to decreased DNA synthesis as measured by [methyl-3H]dTTP incorporation. M13 mp19 single-stranded DNA template modified with 2BA was used to determine the sites of termination of DNA replication by T7 polymerase and Klenow fragment of Escherichia coli DNA polymerase I. It was found that DNA replication stopped frequently before and occasionally opposite deoxycytidine nucleotides. These results indicated that an as yet unidentified deoxycytidine:2BA adduct may have been formed in the reaction of 2BA with M13 DNA. To investigate if such adducts were formed, we reacted 2BA with deoxycytidine in vitro at pH 4.4, and putative deoxycytidine:2BA adducts were isolated by high-performance liquid chromatography. They were characterized by 1H and 13C nuclear magnetic resonance and with fast atom bombardment mass spectrometry as two diastereomeric 3-bromo-7-(beta-D-deoxyribofuranosyl)- 3,4-dihydro-2-hydroxy-(2H,7H)[1,6-a]pyrimidin-6-one adducts and a 3-bromo-7-(beta-deoxyribofuranosyl)-(4H,7H)-pyrimido[1,6-a]pyrimidin-6 -one adduct. Only the latter adduct, however, was formed in the reaction of 2BA with calf thymus single-stranded DNA in vitro. Tris(2,3-dibromopropyl)phosphate is clastogenic. Because clastogenicity may result from DNA adducts that block replication, the role of the presently identified deoxycytidine adducts of the reaction metabolite 2BA in the clastogenicity of tris(2,3-dibromopropyl)phosphate is discussed.

Formation of thymidine adducts and cross-linking potential of 2-bromoacrolein, a reactive metabolite of tris(2,3-dibromopropyl)phosphate.

DNA-adduct formation by the mutagen 2-bromoacrolein (2BA) with DNA was studied. [3-3H]2BA was reacted with single-stranded (ss) DNA or double-stranded (ds) DNA and subsequently incubated with methoxylamine to convert an unstable 2BA:thymidine adduct (Meerman et al., Cancer Res., 49, 6174-6179, 1989) to a stable product. This product was identified in the study as 3-(2"-hydroxy-3"-methoximpropyl)thymidine (HYMETH) by LC-MS. After extensive purification and enzymatic hydrolysis of modified ssDNA and dsDNA, approximately 5% of the covalently bound activity coeluted with added HYMETH standard in a reverse phase HPLC system. Because the unstable 2BA:thymidine adduct may have the potential to form cross-links, we investigated the reaction of this adduct with various nucleophiles in vitro. A reaction occurred between the adduct and cysteine, but not with lysine or deoxynucleosides. Reaction of 2BA with ssDNA in the presence of [3H]glutathione also resulted in the binding of radiolabelled GSH to DNA. These results indicate that the reactive aldehyde group of the adduct can react with thiol groups in proteins to form protein-DNA cross-links. Further, the possibility that tris- and bis-(2,3-dibromopropyl)phosphate (Tris-BP and Bis-BP) form such cross-links was examined in vivo in Drosophila. The results indicate that Tris-BP is a cross-linking agent, whereas Bis-BP is not. Inasmuch as Tris-BP is known to be metabolized rapidly to 2BA and Bis-BP, whereas Bis-BP forms 2BA only very slowly, suggests that in Drosophila DNA adducts are formed that cross-link proteins.