Bruna Bonora

The different genotoxicity of P-dichlorobenzene in mouse and rat: Measurement of the in vivo and in vitro covalent interaction with nucleic acids

Twenty-two hours after i.p. injection to male Wistar rats and BALB/c mice para-dichlorobenzene (p-DCB) is bound covalently to DNA from liver, kidney, lung and stomach of mice but not of rats. DNA adducts in mouse liver are repaired in seventy-two hours. The covalent binding index value, calculated on the labelling of mouse liver DNA, classifies p-DCB as a weak initiator with an oncogenic activity lower than that of chlorobenzene. The labelling of RNA and proteins from the different organs of both species is, however, low. In vitro interaction with calf thymus DNA mediated by mouse and rat microsomes from liver and lung did occur. Binding extent was strongly reduced by addition of 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF 525-A) to the microsomal standard incubation mixture, whereas it was enhanced by adding GSH. Cytosolic fractions from kidney and lung were able to induce binding of p-DCB to DNA to a lower extent with respect to microsome-mediated binding. These results indicate that microsomal mixed function oxidase system and microsomal GSH-transferases can be involved in overall activating metabolism whereas cytosolic GSH-transferases play a minor role. This study, which is a part of a structure-activity relationship approach on benzene and its haloderivatives, provides the first evidence of genotoxicity of p-DCB in mammalian cell. It allows to partly explain variations of susceptibility of different species to hepatocarcinogenesis and of hepatotoxicity of different isomers.

Chloroform bioactivation leading to nucleic acids binding.

Chloroform was bound covalently to DNA, RNA and proteins of rat and mouse organs in vivo after i.p. injection. Covalent Binding Index values of rat and mouse liver DNA classify chloroform as a weak initiator. Labelings of RNA and proteins from various organs of both species were higher than that of DNA. In an in vitro cell-free system, chloroform was bioactivated by cytochrome P450-dependent microsomal fractions, by cytosolic GSH-transferases from rat and mouse liver, and particularly by the latter enzymes from mouse lung. This observation suggests that GSH plays a role In the binding of chloroform metabolites to DNA. The presence of both microsomal and cytosolic enzymatic systems in the standard incubation mixture generally led to an additive or synergistic bioactivating effect for rat and mouse, respectively.

In Vivo and in Vitro Interaction of 1,2-Dichlorobenzene with Nucleic Acids and Proteins of Mice and Rats

Twenty-two hours after i.p. injection into male Wistar rats and BALB/c mice, 1,2-dichlorobenzene (1,2-DCB) was covalently bound to DNA, RNA, and proteins of liver, kidney, lung and stomach. The covalent binding index to liver DNA was typical of carcinogens classified as weak initiators. The enzyme-mediated in vitro interaction of 1,2-DCB with calf thymus DNA or synthetic polyribonucleotides was carried out by a microsomal mixed-function oxidase system and microsomal GSH-transferases, which seemed to be effective only in liver and lung of rat and mouse. Cytosolic GSH-transferases played a minor role in 1-2-DCB bioactivation. The latter finding provides the first evidence of 1,2-DCB genotoxicity in mammalian cells. The type of halide, the number of halosubsti-tuents and their spatial dispostion on the benzene ring are the major determinants of halobenzenes activability to intermediate(s) capable of interacting covalently with DNA and other macromolecules in biologic systems.

GENOTOXICITY OF CHLOROETHANES AND STRUCTURE-ACTIVITY RELATIONSHIPS

Chloroethanes are widely produced and utilized compounds and have extensive human exposure. All of them are recognized as being toxic causing damage to the major parenchymous tissues, such as liver, kidney and central nervous system1. Some of them exert mutagenic activity in short-term tests in vitro but their carcinogenicity has not always been adequately evidenced2,3. These compounds require bioactivation to the proximate toxin or carcinogen via metabolism by cytochrome P450-dependent enzymatic systems forming adducts with the genetic material. In some instances, metabolism results in detoxication or inactivation of the bioactive metabolite to innocuous compound, generally through conjugation with GSH4. The aim of this study is to better identify the genotoxic activity of chloroethanes in in vivo and in vitro systems, also in an attempt to validate a cell-free system as a short-term test for carcinogenicity prediction of initiating compounds, and to find structure-activity relationships among compounds belonging to the same chemical class.

Covalent binding of 1,1,1,2‐tetrachloroethane to nucleic acids as evidence of genotoxic activity

Twenty-two hours after ip administration to male Wistar rats and BALB/c mice, 1,1,1,2-tetrachloroethane (1,1,1,2-TTCE) is bound covalently to DNA, RNA, and proteins of liver, lung, kidney, and stomach. The in vivo reactivity leads to binding values to DNA generally higher in mouse organs than in rat organs. The covalent binding index (CBI) values (82 in mouse liver DNA and 40 in rat liver DNA) classify 1,1,1,2-TTCE as a weak to moderate initiator. Both microsomal and cytosolic enzymatic systems from rat and mouse organs are capable of bioactivating 1,1,1,2-TTCE in vitro. Liver fractions are the most effective. When the activating systems are simultaneously present in the incubation mixture a synergistic effect is observed. Unlike the related chemical 1,1,2,2-tetrachloroethane (1,1,2,2-TTCE), which is bioactivated only through an oxidative route, 1,1,1,2-TTCE metabolism is carried on by oxidative and reductive pathways, both dependent on cytochrome P-450. 1,1,1,2-TTCE is also bioactivated by microsomal GSH-transferases from liver and lung. These data further confirm that correlations exist between structure and genotoxic activity of halocompounds.

The covalent interaction of 1,4-dibromobenzene with rat and mouse nucleic acids: in vivo and in vitro studies

1,4-Dibromobenzene (1,4-DBB) was covalently bound to DNA from liver, kidney, lung and stomach of mice after intraperitoneal administration. The covalent binding index (CBI) value (23 in mouse liver) was typical of weak initiators. On the contrary, no interaction with DNA from rat organs was observed (CBI detection limit: 1.3-2.6). The in vitro interaction of 1,4-DBB with calf thymus DNA was mediated mainly by microsomes, especially those from liver of both species and from mouse lung. Mouse subcellular fractions were more active then rat subcellular fractions. Unlike liver cytosol, subcellular cytosolic fractions from lung, kidney and stomach were capable of bioactivating 1,4-DBB, although to a lesser extent than liver microsomes. Both cytochrome P-450 and GSH-transferases are involved in 1,4-DBB bioactivation.