Giancarlo Arfellini

In vivo and in vitro binding of benzene to nucleic acids and proteins of various rat and mouse organs.

Benzene binds to macromolecules of various organs in the rat and mouse in vivo. Labelling of RNA and proteins is higher (1 order of magnitude) than DNA labelling, which is low in many organs (liver, spleen, bone marrow and kidney), and negligible in lung; no difference between labelling of rat and mouse organs was found. The covalent binding index (CBI) value was about 10, i.e. typical of genotoxic carcinogens classified as weak initiators. In vitro binding of benzene to nucleic acids and proteins is mediated by hepatic microsomes, but not by microsomes from kidney, spleen and lung, or by cytosol from whatever organ. Nucleic acid binding can be induced by pretreatment with phenobarbitone (PB) and suppressed in the presence of SKF 525-A, of cytosol and/or GSH or of heat-inactivated microsomes. Labelling of exogenous DNA is low and is similar in the presence of rat or mouse microsomes in agreement with the low interaction with DNA measured in vivo.

In vivo and in vitro binding of 1,2-dibromoethane and 1,2-dichloroethane to macromolecules in rat and mouse organs

SummaryThe comparative interaction of equimolar amounts of 1,2-dichloroethane and 1,2-dibromoethane with rat and mouse nucleic acids was studied in both in vivo (liver, lung, kidney and stomach) and in vitro (liver microsomal and/or cytosolic fractions) systems. In vivo, liver and kidney DNA showed the highest labeling, whereas the binding to lung DNA was barely detectable. Dibromoethane was more highly reactive than dichloroethane in both species. With dichloroethane, mouse DNA labeling was higher than rat DNA labeling whatever the organ considered: the opposite was seen for the bioactivation of dibromoethane. RNA and protein labelings were higher than DNA labeling, with no particular pattern in terms of organ or species involvement. In vitro, in addition to a low chemical reactivity towards nucleic acids shown by haloethanes per se, both compounds were bioactivated by either liver microsomes and cytosolic fractions to reactive forms capable of binding to DNA and polynucleotides. UV irradiation did not photoactivate dibromoethane and dichloroethane. The in vitro interaction with DNA mediated by enzymatic fractions was PB-inducible (one order of magnitude, using rat microsomes). In vitro bioactivation of haloethanes was mainly performed by microsomes in the case of dichloroethane and by cytosolic fractions in the case of dibromoethane. When microsomes plus cytosol were used, rat enzymes were more efficient than mouse enzymes in inducing a dibromoethane-DNA interaction: the opposite situation occurred for dichloroethane-DNA interaction, and this is in agreement with the in vivo pattern. In the presence of both metabolic pathways, addition or synergism occurred. Dibromoethane was always more reactive than dichloroethane. An indication of the presence of a microsomal GSH transferase was achieved for the activation of dibromoethane. No preferential binding in vitro to a specific polynucleotide was found. Polynucleotide labeling was higher than (or equal to) DNA binding. The labeling of microsomal RNA and proteins and of cytosolic proteins was many times lower than that of DNA or polynucleotides. The in vivo and in vitro data reported above give an unequivocal indication of the relative reactivity of the haloethanes examined with liver macromolecules from the two species and agree, on the whole, with the relative genotoxicity (DNA repair induction ability, mutagenicity and carcinogenicity) of the chemicals.

In vitro microsome- and cytosol-mediated binding of 1,2-dichloroethane and 1,2-dibromoethane with DNA

Metabolic activation of 1,2-dichloroethane (DCE) and 1,2-dibromoethane (DBE) to forms able to bind covalently with DNA occurs in vitroeither by wat of microsomal or cytosolic pathways. The involvement of these two pathways is variable with respect to species or compound tested. Rat enzymes are generally more efficient than mouse enzymes in bioactivating haloalkanes and DBE is more reactive than DCE. This parallels both the previous report on in vivocomparative interaction and the higher genotoxicity of DBE.

In vivo and in vitro binding of epichlorohydrin to nucleic acids

Epichlorohydrin (EC) binds to macromolecules of biological relevance in vivo: DNA is less labelled than RNA and proteins, rat organs interact more than mouse organs, stomach is the most labelled organ with liver, kidney and lung involved in decreasing order. Based on the Covalent Binding Index (CBI), EC is a weak-moderate oncogen, just as other chlorinated hydrocarbons such as 1,2-dichloroethane and carbon tetrachloride. An interaction of EC with nucleic acids (DNA and polyribonucleotides) occurs also in vitro. It is mediated either by chemical reactivity per se of the molecule (near-UV (NUV) irradiation does not photoactivate EC) and by enzymatic (microsomal and/or cytosolic) fractions, whose relative effectiveness is variable in relation to the organ tested. The best substrates for interaction are poly(G) and poly(A) when using microsomal and cytosolic fractions, respectively, whereas the labelling of double-stranded DNA is always lower. On the whole, the picture of enzyme (microsome + cytosol)-mediated in vitro interaction is similar to the pattern of in vivo binding, with the exception of rat stomach enzymes which are inactive in vitro.

In vivo DNA repair after N-methyl-N-nitrosourea administration to rats of different ages.

DNA repair time-course was studied after injury by N-methyl-N-nitrosourea (MNU) in rat liver cells of animals of different ages and in fetuses using hydroxyurea (HU) as inhibitor of scheduled DNA synthesis. DNA repair was a rapid phenomenon, more so in young adults than in newborns, and was not detectable in fetuses. A correlation seems to exist among organ sensitivity to carcinogen, age of animal and DNA repair.

Interaction of Halocompounds with Nucleic Acids

The binding of epichlorohydrin, 1,2-dichloroethane, 1,2-dibromoethane, chlorobenzene, bromobenzene, and benzene to nucleic acids and proteins of different murine organs was studied in in vivo and in vitro systems. The extent of in vivo enzymatic activation of brominated compounds was higher than that of chlorinated chemicals. Aryl halides were bound mainly to liver DNA whereas interaction of alkyl halides with DNA of liver, kidney, and lung gave rise to similar binding extent. In vitro activation of all chemicals was mediated by microsomal P-450-dependent mixed function oxidase system which is present in rat and mouse liver and, in smaller amount, in mouse lung. Activation of alkyl halides by liver cytosolic GSH-transferases even occurred. The relative reactivity of chemicals in vivo, expressed as Covalent Binding Index (CBI) to rat liver DNA, was: 1,2-dibromoethane > bromobenzene > 1,2-dichloroethane > chlorobenzene > epichlorohydrin > benzene. On the whole, it agreed with in vitro activation of chemicals, with genotoxicity data from other short-term assays and also with oncogenicity of benzene, epichlorohydrin, 1,2-dichloroethane, and 1,2-dibromoethane. CBI values of chlorobenzene and bromobenzene gave the first clear evidence of genotoxicity and of possible carcinogenicity of these two chemicals.

The Covalent Binding of Bromobenzene with Nucleic Acids

The hepatotoxic compound bromobenzene binds to DNA, RNA, and proteins of rat and mouse liver in vivo. Binding to a significant extent is also detected in mouse kidney. The covalent binding index (CBI) of bromobenzene is comparable to CBI values of moderately oncogenic substances. The enzyme-mediated in vitro interaction of bromobenzene with calf thyumus DNA and synthetic polyribonucleotides is effected only by microsomes, especially those from mouse and rat liver. Microsomes from mouse lung are also efficient in bioactivating bromobenzene to interact with DNA. Among polyribonucleotides, poly(G) and poly(A) are the most labeled substrates. The suppression of binding to DNA by SKF 525-A and the induction of microsomal activity by a pretreatment with phenobarbitone in vivo confirm that bromobenzene is bioactivated by a P-450 dependent-microsomal mixed function oxidase system. The covalent binding can be the main event to determine the possible carcinogenicity by genotoxic mechanisms. Bromobenzene is photoactivated by ultraviolet light (Λ = 254 nm) to forms capable of interacting with DNA in vitro; the binding is linear up to time.

Comparison Between Photo-Induction and Microsomal Activation of Polycyclic Hydrocarbons with Different Oncogenic Potency

The binding of five polycyclic aromatic hydrocarbons (PAH) (anthracene (A), benzo(a)anthracene (BA), dibenz(a,h)anthracene (DBA), benzo(a)pyrene (BP) and 7,12-dimethylbenz(a)anthracene (DMBA)) to calf thymus DNA and synthetic polyribonucleotides was studied. Binding was mediated by near-ultraviolet (NUV) irradiation and 3-methylcholanthrene-induced microsomes from rat liver, in order to compare the effectiveness of these two activating systems in forming in vitro intermediates capable of binding covalently to nucleic acids. With NUV irradiation, an interaction among PAH and nucleic acids was obtained regardless of the PAH or the nucleic acid employed. The effectiveness of this activating system was higher (between 1 to 2 orders of magnitude) than that shown by induced microsomes. The enzymatic pathway bioactivated all PAH, except A, to interact with DNA. Therefore, a certain degree of correlation between the extent of DNA binding and oncogenic potency of the chemicals seemed to exist. Polynucleotide labeling was always higher than DNA labeling.

PHOTOINDUCED REACTION OF DIMETHYLNITROSAMINE WITH DNA AND POLYNUCLEOTIDES

Abstract— The interaction between products from dimethylnitrosamine photolytic breakdown and DNA is time‐dependent and the reaction with polyguanylic acid is 36 times higher than that occurring with other polyribonucleotides; such interactions occur only if nucleic acids are present during dimethylnitrosamine photolytic breakdown. Among interaction compounds, neither O6‐methyl‐ nor 7‐methylguanine are observable; on the contrary, N‐methylhydrazine and trimethylamine are always found in acid and alkaline hydrolysates of DNA and polynucleotides. These findings are very similar to the results previously found in biological in vivo and in vitro systems and suggest a possible usefulness of this physicochemical system as an analogical model of chemical carcinogenesis.

Removal of 5-bromo-2-deoxyuridine incorporated in liver DNA of newborn and young adult rats.

5-bromo-2-deoxyuridine (BUdR) removal from liver DNA in newborn and young adult rats has been demonstrated: it forms the basis of a reliable method to measure DNA repair in tissues provided with detectable DNA synthetic activity.