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Cancer Letters, 28 (1985) 159--168 Elsevier Scientific Publishers Ireland Ltd. 159 IN VIVO AND IN VITRO BINDING OF BENZENE TO NUCLEIC ACIDS AND PROTEINS OF VARIOUS RAT AND MOUSE ORGANS GIANCARLO ARFELLINI, SANDRO GRILLI, ANNAMARLA COLACCI, MARIO MAZZULLO and GIORGIO PRODI Centro di Cancerogenesi Chimica, Istituto di Cancerologia, Universita degli Studi di Bologna, 40126 Bologna (Italy) (Received 24 December 1984) (Accepted 29 May 1985) SUMMARY Benzene binds to macromolecules of various organs in the rat and mouse T jjaocuiiig ui ivnn anu piot/umo 10 uiguci111 VIVUc /if D \T A nvt/1 /vimp -In VirtVi av ( 1 av a I- w* n rfv\ ifnrl a \ V1UC1 Ol UiaglllLUUU) 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. INTRODUCTION Benzene is extensively produced by the chemical industry (over 15 million tons per year) and widely used as a chemical intermediate for producing pesticides, dyes, plastic resins and commercial gasoline [14]. Nowadays benzene is an environmental pollutant (air, water, food, feed, tobacco, pyrolysis products): the daily inhalation for man in the general urban atmosphere is about 0.6 mg. Its discharge into the atmosphere is regulated in some countries [33]; the permissible level of occupational exposure has been established in most countries as the time-weighted averflcre concen tration of 10 ppm [14]. Benzene is myelotoxic and metabolized by micro somes to epoxide and phenol derivatives [18,19,31] including free radical 0304-3835/85/$03.30 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland derivatives [16] which are all able to interact with nucleophilic centers of proteins [16,18,31]. The evidence for benzene genotoxicity in various experimental systems is contradictory. Benzene induces neither mutagenic effects nor unscheduled DNA synthesis but gives rise to chromosomal aberrations and increases the frequency of sister-chromatid-exchanges and of micronuclei. The agent is embryotoxic but is neither embryolethal nor teratogenic. Myelotoxicity and chromosomal aberrations have also been demonstrated in man [14]. Benzene induces carcinomas of the Zymbal gland and oral cavity in rats after intragastric administration, and, after inhalation, a very low occurrence of hepatocarcinomas, along with liver hyperplasia and dysplasia [23]. Nevertheless, evidence for carcinogenicity in rodents is limited when the incompleteness of experimental data as regards different species, sexes, routes of administration and the conflicting findings on leukemia induction in mice [14] are considered. On the contrary, there is sufficient evidence for leukemia induction in man. The covalent binding index (CBI) could be used as a short-term assay of carcinogenicity for suspected initiators or to compare the carcinogenic potency of different chemicals proven to exert an oncogenic effect [20], thus providing information about the mechanism of action (initiation and promotion). Here, we aimed at obtaining such information by measuring in vivo and in vitro binding of benzene to nucleic acids of various murine organs. MATERIALS AND METHODS Radiochemicals and chemicals [U-14C] Benzene (99.8 mCi/mmol; radiochemical purity 99.5%; chemical purity 99%) was purchased from The Radiochemical Centre, Amersham, U.K. and its specific activity was made 20 mCi/mmol by adding cold benzene to the tracer. DNA, polynucleotides, /3-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and glutathione, reduced form (GSH), were purcahsed from Sigma Chemical Co., St. Louis, MO, U.S.A.; SKF 525-A was a gift from Smith, Kline and French, Welwyn, U.K.; PB was obtained from Carlo Erba, Milan, Italy; lumasolve from Lumac, Basel, Switzerland; readysolve MP from Beckman, Milan, Italy. Other chemicals, all of analytical grade, were obtained from Merck, Darmstadt, F.R.G. In vivo binding to DNA, RNA and proteins Four male Wistar rats (~250 g) and 12 male BALB/c mice (25 g) received i.p. 127 /zCi (6.35 jumol)/kg body wt [14C]benzene dissolved in 0.5 ml (for rats) and 0.1 ml (for mice) sterile 0.9% NaCl solution containing ethanol which was administered at the dosage of 105 mg/kg body wt. Animals were kept without food and killed 22 h after injection. In order to obtain DNA, RNA and proteins, kidneys, lungs and representative aliquots of liver homo 161 genates were pooled and processed according to a phenol method [24]. The same technique was performed on either single rat liver homogenates or on 6 different mouse liver homogenates (each pooling aliquots of homo genates from 2 individual organs) to obtain an estimation of individual varia bility. Fractions were exhaustively washed with organic solvents until no radioactivity was extracted from macromolecules whose recovery, purity and labelling was measured as described elsewhere [24]. In the case of bone marrow, obtained by the method of Gill et al. [7], and mouse spleen, due to limited tissue availability, only DNA labelling was measured. Cells were lysed with 4.4 ml 0.1 M Tris-0.1 M KC1-1 mM EDTA (pH 8)--1% SDS at 0C for 15 min and subjected to CsCl sedimentation as reported elsewhere [2]: the same procedure was performed on aliquots of pooled rat spleen to compare DNA labellings obtained with the two methods described. Organs were processed immediately or stored at -- 30C for a maximum of 1 month. In vitro interaction with nucleic acids and proteins Microsomes and cytosol were obtained according to Ref. 24 from the pooled liver, lungs, kidneys and spleen of animals which received [12] phenobarbitone (PB) (100 mg/kg body wt per day, i.p., dissolved in sterile 0.9% NaCl solution, 0.5 ml for rat (11 animals, ~300 g) and 0.2 ml for mouse (67 animals, *-25 g), during the 2 days prior to killing in order to induce enzymatic activity [29]; 4 rats and 15 mice received no PB. Enzy matic fractions were stored at -- 35C for no more than 1 month. Standard incubation mixture consisted of 2.5 pCi [I4C] benzene in 6.25 pi ethanol, 1.5 mg calf thymus DNA or polynucleotide, 2 mg PB-induced microsomal protein + NADPH (0.8 mM) or 6 mg PB-induced cytosolic protein + GSH (10 mM) to a final volume of 3 ml 0.08 M potassium phosphate--5 mM MgCl2 buffer (pH 7.7). Incubation was carried out in triplicate at 37C in air for 60 min under shaking in the dark. Blanks were performed in the absence of enzymes. The influence on microsome-mediated binding extent of various parameters including non-induced enzymes, time-course (5-, 10-, 30-min incubation), concentration of benzene (1.25 pCi and 5 pCi), addition of 6 mg cytosolic protein and/or GSH (10 mM) to microsomal system, were also tested. In some trials, further blanks (zero time incubation, enzymes but no cofactors, enzymes inactivated with heat at 100C for 10 min) were also carried out. Reaction was stopped by chilling at --20C, microsomes (when present) were separated by ultracentrifugation at 105,000 X g for 1 h at 0C and proteins were removed from the aqueous layer by phenol extraction. For re-isolation of macromolecules (DNA or polynucleotides, microsomal RNA and proteins, cytosolic proteins) see Ref. 24. The statis tical evaluation was performed with Student's f-test. RESULTS Benzene binds to DNA, RNA and proteins of rat and mouse organs in vivo, after administration of a dosage which is nearly 6000-fold lower than the LDS0 for rat (Table 1). Labelling of RNA and proteins is higher than also evaluated: such values are shown as mean S.E. The binding data collected in v itro are m ediated b y PB-induced microsomes from the same organs o f th e 2 species, except fo r bone m arrow , under standard procedure (60-m in incubation at 37C in air, in the dark). Generally, they are reported as mean S.E. o f 3 values (8 in th e case o f rat liver, 5 in the case o f mouse liver) o f to ta l binding: control experiments (blanks) have been systematically perform ed in the absence o f enzymes. CO j .s oL *5"> 4J oo is lH a c-- o rH Tp LA eo 00 CD <* eo rH +i +1 +1 4! C9 Tp rH [> CO Tp LA 0A CD LA LA Tp Q 09 LA tA 09 09 z CO 09 CO 09 09 *! 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So. 2^ 41 2 C rH J--c3o> i^--i z Qh 56D "60-f Ea g> ^ Tsf03l 22rH ^M&ycc .rf5Ce lC C0O) <N CO c cd ^-v o CO yM 0 W> .3 yy0a Oh Q<2 'a 163 164 DNA labelling and similar in the different organs of both species. DNA labelling is low and comparable in liver, spleen and kidney (1 adduct/107 nucleotides is formed in mouse liver DNA) and negligible in mouse lung. Moreover, there is agreement either between phenol extraction and CsCl sedimentation in measuring DNA labelling of rat spleen and between label ling of liver DNA from pooled organs and the mean value of single labelling determinations. Liver microsomes bioactivate benzene to forms capable of interacting with exogenous DNA and microsomal macromolecular constitutents. Binding increases linearly with time up to 30 min, then the increase is sublinear up to 60 min, the incubation time chosen as the standard procedure. Binding extent is directly related to the concentration of tracer (data not shown). When using PB-induced microsomes from various organs (the same as those examined in vivo except for bone marrow), a statistically significant binding to DNA (total binding-blanks) is mediated by liver microsomes alone: no difference between the activity of rat and mouse enzymes is evidenced. Such binding, as well as that to microsomal RNA and proteins, is practically suppressed by SKF 525-A, a potent inhibitor of hepatic microsomal oxida tion [8], by pre-heating of microsomal fractions or when cofactors are omitted. A very low chemical reactivity per se of benzene towards macro molecules is, however, observable in blank experiments. Furthermore, PB /-s Poly(A) Poly(G) Poly(U) Poly(C) Fig. 1. In vitro binding of benzene to synthetic polynucleotides mediated by PB-induced hepatic microsomes of rat and mouse under standard incubation conditions (see Table 1). Data from triplicate experiments are reported as percentage (mean S.E.) of the binding to poly(A) mediated by rat microsomes taken as 100 (5.17 1.84 pmol/mg). , blank values obtained in the absence of enzymes. Labelling of rat liver microsomal RNA and proteins, given for example in the case of interaction with poly(A), is: 37.93 7.52 and 464.36 118.91 pmol/mg, respectively. 165 induced-microsomes are twofold more active than normal microsomes in mediating an interaction between benzene and DNA, whereas the addition of cytosol and/or GSH to the microsomal system strongly inhibits the extent of binding. Cytosol is not capable of mediating a statistically significant interaction with macromolecules whatever the organ tested (data not shown). The pattern of interaction with polyribonucleotides mediated by liver microsomes is shown in Fig. 1. Labelling decreases in the following order: poly(A) > poly(G) > poly(U) > poly(C) and is lower than DNA labelling measured under identical conditions: nevertheless, the opposite situation is observed, regardless of polynucleotide considered, with labelling of microsomal RNA and proteins, an example of which is given in the legend of Fig. 1. DISCUSSION The covalent binding index (CBI) of benzene is of the same order as that found with other chemicals defined as weak carcinogens (Table 2) and lower TABLE 2 BINDING OF SOME HEPATOCARCINOGENS TO RAT LIVER DNA IN TERMS OF CBI Oncogen Reference CBIa Oncogenic potency Aflatoxin B, Dimethylnitrosamine Aflatoxin M, [5] [9] [20] 17,000 2310 1600 Strong 2 -Acety laminofluorene Vinyl chloride 1,2-Dibromoethaneb o-Aminoazotoluene [10] [3] [1] [17] 560 525 515 Moderate 230 1,2 -Dichloroethane Carbon tetrachloride Urethan Epichlorohydrinb [1] [27] [26] [24] 47 46c 34 23 a -Hexachlorocyclohexane Saccharin*1 [28] [22] <0.1 Cocarcinogen or <0.005 promoter aClassification of CBI values with respect to oncogenic potency [20]: in the thousands, strong; in the hundreds, moderate; in the tens, weak for initiators; < 1 non-genotoxic oncogens (promoters and cocarcinogens). bThe hepatocarcinogenic effect of such oncogenic chemicals has not been proven up to now. c Mouse liver instead of rat liver. dNon-hepatocarcinogen capable of inducing bladder tumors (CBI value in bladder DNA < 0.05). than that found with its halogenated derivatives bromobenzene (CBI = 255 and 225 for rat and mouse liver, respectively) (unpublished data) and chloro benzene (CBI= 38 and 36 for rat and mouse liver, respectively) (unpublished data). The CBI value reported here for benzene agrees with the previous report of Lutz and Schlatter [21] concerning only liver DNA from rats exposed to the chemical by inhalation; it is higher than that typical of promoters but many times lower than that of such potent carcinogens as aflatoxin Bi (Table 2). We are aware of another study on benzene interaction with nucleic acids in vivo [6] which is, however, not comparable to the present study since no attempt to separate DNA from RNA in the nucleic acid fraction and purify them from protein contamination was performed. The enzyme-mediated binding of benzene to nucleic acids in vitro is per formed by liver microsomes alone: binding extent is low and inferior to that found with chlorobenzene (2-fold) and bromobenzene (4-fold) (unpublished data) under identical experimental conditions. On the other hand, the microsome-mediated binding we found is similar to that previously reported [16,31], measuring a 10-fold lower binding to microsomal proteins alone by incubating a 7-fold inferior concentration of benzene. Purine polynucleo tides are better binding acceptors than pyrimidine polynucleotides: labelling is unexpectedly lower than DNA labelling, unlike previous reports for different chemicals [1,24]. Benzene is bioactivated by microsomal mixed function oxidase system; the activation of benzene occurs mainly in liver and, possibly, in bone marrow [15]. The active intermediate formed in liver should be stable enough to exert its effect at a distance as shown by labelling of proteins in various organs. Similar to the metabolic activation of bromo benzene and chlorobenzene [32, unpublished data], the phenolic derivative and p-benzoquinone are the benzene intermediates most probably involved in the interaction with DNA which is suppressed by GSH [11]. Phenol is originated either from benzene oxide [18] or from phenolic radical [16] whereas p-benzoquinone is generated from phenol [19]. Both metabolites covalently bind to cellular macromolecules: the conjugation of p-benzo quinone with GSH is predominantly a non-enzymatic process [19]. On the whole, both in vivo and in vitro interactions give evidence for a weak initiating activity of benzene: this agrees with results from other short-term assays of genotoxicity [14] which are partly contradictory. It should be pointed out that, in order to evaluate the carcinogenicity of chemicals, all short-term data, chemical properties, metabolism and mecha nism of action should be considered along with data from long-term assay [13], collected in both sexes of different species, in different experiments by various routes of administration. Moreover, the statistical evaluation we performed with Fisher exact and Cochran-Armitage tests on data from a recent long-term assay [23] showed that the dosage inducing a significant occurrence of carcinomas of Zymbal gland and oral cavity in both female and male rats (500 mg/kg per day) is many fold higher than that adminis tered when dealing with such potent carcinogens as aflatoxins, dimethyl- ID / nitrosamine, 7,12-dimethylbenz [a] anthracene and 4-nitroquinoline-l-oxide. Therefore, the results of long-term assays performed with benzene[14,23] are not yet satisfactory. Nevertheless, benzene carcinogenicity in humans is sufficiently proven and differences in organ susceptibility between rodents and man seem to exist. In our opinion, what needs to be done in the future is: (a) to test the initiating and/or promoting effects of benzene in liver model systems of Farber [4] and Peraino [25] on female rats which develop liver hyperplasia and dysplasia and present a very low incidence of liver tumors [23]; (b) to collect further information on the adducts with DNA: however, strong limitations in performing such a study are due to the extremely low specific activities of nucleic acids achieved in in vitro systems. ACKNOWLEDGEMENTS This work was supported in part by grants from "Associazione Itaiiana per la Ricerca sul Cancro", Milan and from Ministero della Pubblica Istruzione, Rome, Italy. REFERENCES 1 Arfellini, G., Bartoli, S., Colacci, A., Mazzullo, M., Galli, M.C., Prodi, G. and Grilli, S. (1984) In vivo and in vitro binding of 1,2-dibromoethane and 1,2-dichloroethane to macromolecules in rat and mouse organs. J. Cancer Res. Clin. Oncol., 108, 204--213. 2 Arfellini, G., Grilli, S. and Prodi, G. 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