Gisèle Brun

Evaluation of the genetic and embryotoxic effects of bis(tri-n-butyltin―oxide (TBTO), a broad-spectrum pesticide, in multiple in vivo and in vitro short-term tests

The genetic and embryotoxic effects of bis(tri-n-butyltin)oxide (TBTO) were evaluated in multiple in vivo and in vitro short-term tests preparatory to its potential wide use as a molluscicide in control of schistosomiasis. When tested in the rec assay in Bacillus subtilis, TBTO was not mutagenic and it did not induce reverse mutations in Klebsiella pneumoniae. Neither in the presence nor in the absecne of rat liver activation system did TBTO produce point mutations in Salmonella typhimurium strains TA1530, TA1535, TA1538, TA97, TA98 or TA100. TBTO was matagenic in strain TA100 in a fluctuation test, but only in the presence of rat liver S9 (Aroclor-induced). TBTO did not induce gene mutations in the yeast Schizosaccharomyces pombe, mitotic gene conversions in the yeast Saccharomyces cerevisiae, nor sister-chromatid exchange in Chinese hamster ovary cells in the presence or absence of rat or mouse liver S9. In the latter cells, structural chromosomal aberrations, endoreduplicated and polyploid cells were induced. TBTO did not induce gene mutations in V79 Chinese hamster cells (to 8-azaguanine-, ouabain- or 6-thioguanine-resistance) in the presence of a rat liver postmitochondrial fraction or in cell (hamster embryo cells and human and mouse epidermal keratinocyte)-mediated assays. In mouse lymphoma cells, TBTO did not induce 6-thioguanine- or BUdR-resistant mutations. As many tumour promoters inhibit metabolic cooperation between V79 Chinese hamster 6-thioguanine-resistant/-sensitive cells, TBTO was tested but showed no such activity. TBTO was examined for the induction of recessive lethal mutations in adult Berlin K male Drosophila melanogaster, either by feeding or by injection. Doses of 0.37 or 0.74 mM did not increase the number of X-linked recessive lethal mutations. An increased number of micronuclei was observed in the polychromatic erythrocytes of male BALB/c mice 48 h after a single oral dose of TBTO (60 mg/kg bw), while a lower dose (30 mg/kg bw) was ineffective. Neither of the two doses had induced micronuclei 30 h after treatment. The reproductive toxicity of TBTO was studied in NMRI mice. In a 10-day toxicity study, the LD50 and LD10 were 74 and 34 mg/kg bw, respectively. An increased frequency of cleft palates was seen in the fetuses of mice (compared with controls, 0.7%) treated orally during pregnancy with 11.7 mg/kg TBTO (7%), 23.4 mg/kg (24%) or 35 mg/kg (48%).(ABSTRACT TRUNCATED AT 400 WORDS)

Some factors determining the concentration of liver proteins for optimal mutagenicity of chemicals in the Salmonella/microsome assay.

In plate assays in the presence of S. typhimurium TA100 and various amounts of liver 9000 X g supernatant (S9) from either untreated, phenobarbitone- (PB) or Aroclor-treated rats, the S9 concentration required for optimal mutagenicity of aflatoxin B1 (AFB) depended both on the source of S9 and on the concentration of the test compound. In these assays, the water-soluble procarcinogen, dimethylnitrosamine (DMN) was mutagenic in S. typhimurium TA1530 only in the presence of a 35-fold higher concentration of liver S9 from PB-treated rats than that required for AFB, a lipophilic compound. In liquid assays, a biphasic relationship was observed in the mutagenicities in S. typhimurium TA100 of benzo[a]pyrene (BP) and AFB and the concentration of liver S9. For optimal mutagenesis of BP, the concentration of liver S9 from rats treated with methylcholanthrene (MC) was 4.4% (v/v); for AFB it was 2.2% (v/v) liver S9 from either Aroclor-treated or untreated rats. At higher concentrations of S9 the mutagenicity of BP and of AFB was related inversely to the amount of S9 per assay. The effect of Aroclor treatment on the microsomemediated mutagenicity of AFB was assay-dependent: in the liquid assay, AFB mutagenicity was decreased, whereas in the plate assay it did not change or was increased. As virtually no bacteria-bound microsomes were detected by electron microscopy, after the bacteria had been incubated in a medium containing 1-34% (v/v) MC-treated rat-liver S9, it is concluded that, in mutagenicity assays, mutagenic metabolites generated by microsomal enzymes from certain pro-carcinogens have to diffuse through the assay medium before reaching the bacteria. Thus the mutagenicity of BP was dependent on both the concentration of rat-liver microsomes and that of total cytosolic proteins and other soluble nucleophiles such as glutathione. At a concentration of 4.4% (v/v) liver S9, the mutagenicity of BP was about 3.6 times higher than in assays containing a 4-fold higher concentration of cytosolic fraction. Studies on the glutathione-dependent reduction of BP mutagenicity in plate assays has shown that, in the presence of liver S9 concentrations greater than that required for optimal mutagenicity, the reduction in mutagenicity was related directly to the concentration of liver S9. Thus, in the Salmonella/microsome assay, when the concentration of rat-liver S9 was increased over and above the amount required for the optimal mutagenicity of BP, the mutagenic metabolites of BP were inactivated (by being trapped with cytosolic nucleophiles and/or by enzymic conjugation with glutathione); this effect increased more rapidly than their rate of formation. The concentration of liver S9 for optimal mutagenicity of test compounds requiring activation catalyzed by mono-oxygenases seems, therefore, to be related to the departure from linearity of the relationship between the rate of formation of mutagenic metabolites and the concentration of liver S9.

Structure-activity studies in E. coli strains on ochratoxin A (OTA) and its analogues implicate a genotoxic free radical and a cytotoxic thiol derivative as reactive metabolites ☆

Ochratoxin A (OTA), its major metabolite in rodents, ochratoxin alpha, and seven structurally related substances were assayed for SOS DNA repair inducing activity in Escherichia coli strain PQ37. At concentrations of 0.1-4 mM, OTA, chloroxine, 5-chloro-8-quinolinol, 4-chloro-meta-cresol and chloroxylenol induced SOS DNA repair in the absence of an exogenous metabolic activation system. Ochratoxin B, ochratoxin alpha, 5-chlorosalicylic acid and citrinin were inactive, but all except ochratoxin alpha were cytotoxic. Thus, the presence of chlorine at C-5 appears to be one determinant of genotoxicity in these substances. Amino oxyacetic acid, an inhibitor of the cysteine conjugate beta-lyase, decreased the cytotoxicity of OTA but did not alter its genotoxic activity, suggesting the formation of a cytotoxic thiol-containing derivative. The mechanisms by which OTA and some of its active analogues induce SOS DNA repair activity was further investigated in E. coli PQ37 and in three derived strains (PQ300, OG100 and OG400), containing deletions within the oxy R regulon. The response in strain PQ37 was measured in the absence and presence of Trolox C, a water-soluble form of vitamin E. Trolox C completely quenched the genotoxicity of OTA, and the effect was similar in the mutant and wild-type strains. These results implicate an OTA-derived free radical rather than reduced oxygen species as genotoxic intermediate(s) in bacteria.

Some aspects of metabolic activation of chemical carcinogens in relation to their organ specificity

The role of organ-specific, enzymic release of alkylating intermediates in determining which tissue develops a tumour in response to a given N-nitrosamine has been evaluated on the basis of published data on the carcinogenicity of 62 N-nitrosamines that induce tumours in specific tissues in rats. A good correlation was noted between the metabolic capacity for N-nitrosamine activation and the organ in which tumours are induced. A relationship was also noted between the localization of carcinogen activating enzymes in rat tissues and the site at which the tumour developed following administration of N-(acetoxy) methyl-N-methylnitrosamine. This compound was shown to be cleaved by soluble enzymes equally efficiently in various rat tissues, such as liver, kidney, spleen and small intestinal mucosa, to yield the alkylating and mutagenic intermediates which are presumably those also formed from the parent N,N-dimethylnitrosamine by microsomal enzymes. N-(acetoxy)methyl-N-methylnitrosamine causes tumours of the gastrointestinal tract, although the parent N,N-dimethylnitrosamine rarely affects this site in rats.The neurotropic carcinogen 3,3-dimethyl-1-phenyltriazene is known to undergo predominantly in the rodent liver oxidative N-mono-demethylation by microsomal enzymes to yield a carcinogenic, mutagenic, and alkylating intermediate, 3-methyl-1-phenyltriazene; however the parent compound produces extrahepatic tumours exclusively. To explain this alternative model of organ specificity, the half-life of 3-methyl-1-phenyltriazene was measured and found to be long enough to permit its distribution throughout the body. Furthermore, subcutaneous injection of 3-[C14-methyl]-1-phenyltriazene into rats yielded alkylated bases in nucleic acids of hepatic and extrahepatic tissues including brain, the major target organ of the parent compound 3,3-dimethyl-1-phenyltriazene. Eight hours after injection of 3-methyl-1-phenyltriazene the O6 ∶ N7-methylguanine ratio was found to be lowest in the liver and highest in the brain, indicating a low rate of O6-methylguanine excision. Thus, for this carcinogen, the persistence of alkylated DNA bases may be a final determinant in tissue specific induction of tumours.The implications of these data for the use of in vitro metabolic activation systems in short-term tests for detecting potential carcinogens are discussed.ZusammenfassungDie Rolle einer organspezifischen Freisetzung von alkylierenden Zwischenstufen aus N-Nitrosaminen in bezug auf ihre organotrope, karzinogene Wirkung, wurde anhand von Literaturdaten an 62 N-Nitrosaminen untersucht. Die Fähigkeit verschiedener Gewebe der Ratte, N-Nitrosamine metabolisch zu aktivieren, ließ sich gut mit Organen korrelieren, in denen nach Vergabe der N-Nitrosamine Tumoren erzeugt werden.Ein ähnlicher Zusammenhang wurde beim Karzinogen N-(Acetoxymethyl)-N-methylnitrosamin beobachtet. Diese Nitrosoverbindung wurde durch lösliche Enzyme, die in gleich hoher Aktivität in der Leber, Niere, Milz und im mukösen Gewebe des Dünndarmes vorkommen, in alkylierende und mutagene Zwischenstufen gespalten, wobei wahrscheinlich die gleichen reaktiven Metaboliten freigesetzt werden, wie sie aus dem hepatokarzinogenen N,N-Dimethylnitrosamin durch mikrosomale Enzyme gebildet werden. Im Gegensatz zur letzteren Substanz, induziert jedoch N-(Acetoxy)methyl-N-methylnitrosamin bevorzugt Tumoren im Verdauungstrakt der Ratte.3,3-Dimethyl-1-phenyltriazen, ein Karzinogen mit neurotroper Wirkung, wird hauptsächlich in der Leber von Nagetieren über eine oxidative N-Monodemethylierung in das karzinogene, direkt mutagene und alkylierende 3-Methyl-1-phenyltriazen überführt, während die Muttersubstanz überwiegend Tumoren außerhalb der Leber induziert. Die Halbwertzeit von 3-Methyl-1-phenyltriazen wurde in vitro bestimmt und für lang genug befunden, um eine Verteilung dieses Metaboliten im Organismus zu erlauben. Nach subkutaner Vergabe von 3-[14C-methyl]-1-phenyltriazen an Ratten wurden alkylierte Nukleinsäurebasen in der Leber und in anderen Geweben, einschließlich im Hirn, einem Zielorgan des karzinogenen 3,3-Dimethyl-1-phenyltriazens, gefunden wurden. Acht Stunden nach Injektion von 3-Methyl-1-phenyltriazen in Ratten, wurde das O6 ∶ N7-Methylguanin-Verhältnis am niedrigsten in Nukleinsäuren der Leber, aber am größten in denen des Hirngewebes gefunden. Diese Daten unterstreichen eine möglicherweise determinierende Rolle der Halbwertszeit bestimmter alkylierter DNS-Basen in vivo bei der selektiven Erzeugung von Hirntumoren durch 3,3-Dimethyl-1-phenyltriazen.Die Bedeutung dieser Befunde, im Hinblick auf die Benutzung von Organhomogenaten als Aktivierungsystem in Kurzzeit-Testen zur Erfassung potentieller chemischer Karzinogene, wird diskutiert.