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1985 13: 276Toxicol PatholAnnamaria Colacci, Giancarlo Arfellini, Mario Mazzullo, Giorgio Prodi and Sandro Grilli

The Covalent Binding of Bromobenzene with Nucleic Acids  

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T ~ X I C ~ L ~ C I C PATHOLOGY lSSN:0192-6233 Copyright 0 1985 by the Society of Toxicologic Pathologists

Vol. 13, No. 4, 1985 Printed in U.S.A.

The Covalent Binding of Bromobenzene with Nucleic Acids*

ANNAMARIA COLACCI, GIANCARLO ARFELLINI, MARIO MAZZULLO, GIORGIO PRODI, AND

SANDRO GRILLI

Centro di Cancerogenesi Chimica;Zstituto di Cancerologia, Universita di Bologna, 1-40126 Bologna, Italy

ABSTRAO

The hepatotoxic compound bromobenzene binds to DNA, RNA, and proteins of rat and mouse liver in vivo. Binding to a significant extent i s also detected in mouse kidney. The covalent binding index (CBI) of bromobenzene i s comparable to CBI values of moderately oncogenic substances. The enzyme-mediated in vitro interaction of bromobenzene with calf thyumus DNA and synthetic poly- ribonucleotides i s 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 i s 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 i s photoactivated by ultraviolet light (A = 254 nm) to forms capable of interacting with DNA in vitro; the binding i s linear up to time.

I N T R O D U C T I O N Bromobenzene is an environmental con-

taminant known to cause liver necrosis (17, 33). This compound is, not hepatotoxic by itself, but i t must be metabolized to a reactive form, presumably the 3,4-epoxide, capable of binding to liver proteins (17, 26, 41). This reaction is mediated by a microsomal mixed function oxidase via cytochrome P-450 (20). Moreover, a detoxification step has been de- tected through glutathione conjugation (27). However, a simple correlation between hep- atotoxicity of bromobenzene and protein binding did not emerge from all these studies. More recently, Casini et a1 (6) pointed out that lipid peroxidation plays a significant role in determining lethal cell injury and stated that the lipid peroxidation process could be the major cause for liver injury produced by

Address correspondence to: Sandro Grilli, lstituto di Can- ccrologia. Univcrsili di Dolognn. Vialc Filopanti 22. 40126- Bologna. Italy.

bromobenzene administration (7). Neverthe- less, covalent binding remains the widely accepted hypothesis to explain the liver cell necrosis produced by a variety of xenobiotics and the carcinogenicity of many compounds via genotoxic mechanisms. In the present paper, we aimed at detecting bromobenzene genotoxicity utilizing the measurement of its covalent interaction with nucleic acids in in vivo and in vitro systems as a short-term test of carcinogenicity .

METHODS Materials. [U-'4C]Bromobenzene (20 mCi/

mmol; radiochemical purity: >98%) was pur- chased from The Radiochemical Centre, Amersham, UK. DNA, polyribonucleotides, p-nicotinamide adenine dinucleotide phos- phate, reduced form (NADPH), and glutathi- one, reduced form (GSH), were obtained from Sigma; lumasolve from Lumac, Basel, Switz- erland: ready-soh MP from Beckman, Milan, Italy; phenobarbitone (PB) from Carlo Erba,

276

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Vol. 13, No. 4, 1985 BROMOBENZENE BINDING TO DNA 277

Milan, Italy. Other chemicals, all of analytical grade, were obtained from Merck, Darmstadt, Germany.

Animals and Diet. Inbred adult male Wistar rats and BALB/c mice were obtained from Morini, S. Polo D'Enza, Italy and from Charles River, Calco, Italy, respectively. The animals were housed in macrolon cages at 22°C with 12-hr light and 12-hr darkness, receiving a standard pellet diet (Vogt-Moller, Piccioni, Brescia, Italy) and drinking water ad libitum.

In Vivo Studies: Binding to DNA, RNA, and Proteins. Four rats (-250 g) and 1 2 mice (-28 g) received intraperitoneally bromobenzene (127 pCi (6.35 pmol)/kg body weight) dis- solved in dimethyl sulfoxide-sterile 0.9% NaCl solution (5:l). Animals were killed and bled 22 hr after injection. They had been starved all through this period. Livers, kid- neys, and lungs were removed, pooled, and processed as described elsewhere (24) in or- der to measure specific activity of DNA, RNA, and proteins.

In Vitro Studies: Enzyme-mediated Binding. Enzymatic fractions were extracted from rats and mice which had been pretreated with PB (100 mg/kg/day, dissolved in sterile 0.9% NaCl solution injected intraperitoneally) for 2 days prior to death. Microsomes and cyto- sols were obtained and stored as previously described (24). The standard incubation mix- ture consisted of 2.5 pCi bromobenzene, 1.5 mg DNA or polynucleotide, 2 mg microsomal protein plus 2 mg NADPH or 6 mg cytosolic proteins plus 9.2 mg GSH to a final volume of 3 ml 0.08 M potassium phosphate-5 mM MgCl, buffer, pH 7.7. Reactions were carried out in duplicate or triplicate at 37°C in air for 60 min in the dark. The influence of various parameters (noninduced enzymes; time course; concentration of bromobenzene, enzymes, or DNA; addition of 1.5 mM SKF 525-A or of 10 mM GSH and/or cytosolic fractions to microsomal system) on binding extent was also tested. Blanks were system- atically performed in the absence of cofac- tors. As further controls, some blanks were carried out in the absence of enzymes, with heat-inactivated enzymes, or at zero time incubation. Isolation, purification, and label- ing determination of DNA, microsomal RNA, and proteins and of cytosolic proteins were performed as described by Mazzullo et a1 (24).

Photoirradiation. Bromobenzene (12.5 pCi) and calf thymus DNA (7.5 mg) dissolved in 15 ml 0.08 M potassium phosphate buffer (pH

7.8) were poured into a closed spectrophoto- metric, 1-cm thick quartz cell and irradiated for 0, I, 2, and 3 hr at 22 f 02°C in air. Photoirradiation was carried out either with low pressure (NN 15/44, X = 254 nm) or with mean pressure (Q-400, provided with a Sovi- re1 filter: X = 310-395 nm, A,,, = 365 nm) mercury vapor lamps. The incident fluence rate of the beam, measured by ferric oxalate actinometer (4), was 84 J/cm'/min and 1790 J/cm'/min for X = 254 nm and A,,, = 365 nm, respectively. As a further control, a 3-hr in- cubation was performed in the dark under identical experimental conditions. At each time point, 1-ml aliquots were removed from the spectrophotometric quartz cell and DNA labeling was determined as previously de- scribed ( I ) . Assays were always in duplicate.

Statistical Analysis. Differences were ana- lyzed by Student's t test with a level of cbn- fidence of 95%.

RESULTS Table I shows the results obtained in in

vivo interaction studies and the covalent binding index (CBI) values are also reported. Liver DNA labeling is the highest in both species. The binding to macromolecules of mouse kidney is far higher than that to rat kidney macromolecules. Negligible radioac- tivity is associated with lung DNA of both species. RNA labeling of assayed organs is always higher than DNA labeling. Interaction with proteins leads to steady binding values which are lower than RNA labelings and never inferior to those of DNA.

TABLE I-/n Vivo Binding of [U-"C] Bromobenzene to Macromolecules of Various Organs from Rat and Mouse'

Liver Kidney Lung

Rat Mouse Rat Mouse Rat Mouse Macromolecule

DNA 5.25' 4.62 0.58 4.48 0.25 0.07

RNA 9.64 9.10 5.68 19.3 9.00 7.83 Protein 5.00 4.64 3.89 4.63 4.46 3.60

(255)' (225)'

Data refer to pooled organs from 4 male Wistar rats and 12 male BALB/c mice which received 127 pCi/kg body weight of [ U-"Clbromobenzcne 22 hr before kill- ing. Data are expressed in specific activity (picomoles/ mg). ' Corresponding to 233 dpm/mg and to 1.71 pmol/

CBI, calculated according to Lutz (22), is given in mol DNA-P (according to Ref. 40).

parentheses.

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2 78 COLACCI ET AL TOXICOLOGIC PATHOLOGY

Figure 1 shows the time course of micro- some-mediated interactions of bromoben- zene with DNA. Binding increased linearly up to 60 min of incubation and positive cor- relations were found between the extent of binding and the concentration of either tracer or microsomal protein, and an inverse rela- tionship was evident with the amount of DNA acceptor (data not shown). PB is a strong inducer of binding to proteins and RNA (2- Io-fold) and, mainly to DNA (20-fold) (Table 11). Therefore, PB-induced fractions have been systematically employed as the stand- ard procedure. With heat-inactivated micro- somes or in the presence of SKF 525-A, bro- mobenzene binding to DNA is suppressed. Values are similar to those of other blanks (29-185 dpm/mg) and quite comparable to the chemical reactivity of bromobenzene per se (blanks of photoirradiation are reported in Table IV). The addition of GSH or cytosolic enzymes to NADPH-containing standard mi- crosomal system strongly inhibits the binding to DNA catalyzed by rat hepatic microsomes. When only hepatic cytosol is employed, the interaction of bromobenzene with DNA does not occur. Negligible values can be occasion- ally detected and the same phenomenon is observed with lung and kidney cytosols.

90 -~ ~~

3 0 60

Time Cmin)

FIG. 1-Time course of bromobenzene binding to DNA in vifro mediated by PB-induced micro- somes from rat liver. The data are expressed as the mean of duplicate net values, differing in less than 8%, blank values (NADPH-deprived incubations) having always been subtracted from total binding. See text for further explanation.

TABLE Il-fn Vifro Binding of [U-"C] Bromobenzene to DNA Mediated by Hepatic Microsomes'

Incubation Mixture Rat Mouse

dpm/mg Normal microsomes 70 88 Heat-inactivated microsomesb 34 16 PB-induced microsomes:

Standard procedured.' Plus 1.5 mM SKF 525-A 21 27 Plus 10 m M GSH Plus PB-induced cytosold 7 9

1645 k 148 1927 -t 390'

23 1

a Data are reported as means of duplicate or triplicate (k SE) net values: controls (blanks), ranging from 6 5 to 88 dpm/mg, have always been taken off.

PB-induced microsomes were inactivated by heating at 10O.C for 10 min before use.

Corresponding to 43.4 -C 8.78 pmol/mg DNA. dAnimals were pretreated for 2 days prior to death

with 100 mg/kg/phenobarbitone (PB) administered ip. ' Standard incubation procedure: 2.5 pCi [U-"C]bro-

mobenzene. 1.5 mg DNA, 2 mg microsomal protein, and 2 mg NADPH, in 3 ml of 0.08 M potassium phosphate-5 mM MgC12, pH 7.7, were allowed to react in air for 60 min at 37°C in the dark.

Microsome-catalyzed binding of bromo- benzene to DNA is mainly mediated by liver enzymes of both species (Table 111). Mouse microsomes are slightly more efficient than rat microsomes. Also, lung microsomer from mouse are capable of bioactivating bromo- benzene. Labeling of microsomal RNA and proteins is similar to those detected in liver fractions. Rat lung microsomes, as well as mouse and rat kidney microsomes, give rise to a negligible binding to DNA. Labeling of microsomal RNA and proteins is significantly' higher than that of DNA. Nevertheless, the pattern of interactions with microsomal con- stituents in the different organs is similar to that of binding to DNA. Interaction of bro- mobenzene with polyribonucleotides, cata- lyzed by microsomal enzymes from rat and mouse liver, shows a slight preference toward poly(A) and poly(G) (Fig. 2). Polynucleotide labeling is lower than DNA labeling, regard- less of the microsomes employed.

Bromobenzene is photoactivated by irra- diation with X = 254 nm to react with DNA. Binding is near linear up to 3 hours (Table IV). Blanks obtained after 3-hr incubation in the dark and at 0-hr irradiation are low and comparable. No evidence of time-dependent photo-induced binding to DNA is obtained under near-ultraviolet irradiation.

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Vol. 13, No. 4, 1985 BROMOBENZENE BINDING TO DNA 279

TABLE Ill-Microsorne-mediated Binding of [U-"CIBromobenzene to DNA, Microsomal RNA, and Proteins under Standard Incubation Procedureab

Microsomal Microsomal RNA Proteins DNA

Species and Organ

~~ ~~~ ~ ~

dpm/ms Rat

Liver 1,645 f 148 5,380 i- 434 40,394 f 1,745 Kidney 15 f 19' 33 i- 21' 316 & 135 Lung UD' 145 i- 7 500 & 255

Mouse Liver 1,927 i- 390d 4,3 18 -t 488 47,638 f 6,126 Kidney 52 f 33' 155 ? 14 1,157 f 150 Lung 517 f 157 5,804 & 131 46,257 i- 8,121

'See legend of Table I I . Specific activity data are reported as mean i- SE of three net values: control values have been substracted. Blanks: DNA: 85 f 14 (65-88 dpm/mg); RNA: 1 16 & 16 (range: 93- 158 dpmlmg); pro- tein: 564 & 68 (range: 340-827 dpm/mg). ' Nonsignificant differences (p > 0.1) between mean

values from standard tubes and blanks at Student's I test. ' Undetectable.

Corresponding to 43.4 i- 8.78 pmol/mg.

= Mouse

ili 0 Rat

FIG. 2-Microsorne-mediated binding of bro- mobenzene to polyribonucleotides in vilro after 60-min incubation. The data are expressed as the percentage of net binding to poly(A) mediated by rat liver rnicrosornes (given as 100 and correspond- ing to 1281 f 283 dpm/mg) and represent the mean of triplicate experiments SE. See text for further explanation.

DISCUSSION The extent of binding of bromobenzene to

lung and kidney proteins gives evidence for acute toxicity in these organs besides in liver

TABLE IV-Photoinduced in Vitro Binding of [U-'4C]Brornobenzene to DNA'

Wavelength 1 Hr 2 Hr 3 Hr

nm dpm/ms 254 444' 878 1263

(max 365) 191 219 239

Each data point refers l o incubation of 2.5 pCi [U- "C]bromobenzene and 1.5 mg calf thymus DNA in 3 ml 0.08 M potassium phosphate buffer, pH 7.8, and repre- sents mean of two net values, differing from each other in less than 7.0%: zero time irradiation binding values (blanks) (86 and 100 dpm/mg for X = 254 nm and A,, = 365 nm, respectively) have been taken off. The interac- tion occurring after 3-hr incubation in the dark was 146 and 180 dpm/mg for X = 254 nm and A,, = 365 nm, respect ively.

310-395

Corresponding to 10 pmol/mg.

(32,34). However, the chemical binds to DNA in liver more than in lung and kidney after intraperitoneal administration of a dosage which is lower than the minimal hepatotoxic dose of 1.8 mmol/kg (26). The liver seems, therefore, to be a target also for bromoben- zene genotoxicity. The CBI value, calculated on liver DNA labeling, is typical of initiators and higher than the index of cocarcinogens and promoters which are effective through nongenotoxic mechanisms (Table V). More- over, it is similar to the CBI of 1,a-dibromo- ethane (I), a chemical capable of inducing tumors in stomach, lung, spleen, and skin of rats and mice (16, 39), and of oncogenic sub- stances classified as moderately carcinogenic according to Lutz (22) (Table V).

Irreversible binding of bromobenzene to DNA and macromolecular constituents of microsomes takes place in vitro via a P-450 dependent-microsomal mixed function oxi- dasc system. In fact, pretreatment of ani- mals with PB, an inducer of cytochrome P-450, strongly enhances microsome-medi- ated binding. This has been demonstrated previously, with respect to protein binding alone in cell-free preparations (17, 20) and in hepatocytes (6). The formation of bromoben- zene 3.4-oxide is PB inducible and constitutes the intermediate involved in hepatic necrosis (1 8). Pretreatment \v i t h 3 -me thy1 c hol a n t r en e protects against bromobenzene-induced he- patic necrosis via bromobcnzene 2,3-epoxide formation (19). Enzymatic bioactivation of bromobenzene is confirmed by both micro- soma1 inactivation with heating and lack of enzyme-mediated interaction in the presence of SKI: 525-A. The direct relationship be-

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280 COLACCI ET AL TOXICOLOGIC PATHOLOGY

TABLE V--Binding of Hepatocarcinogens to Rat Liver DNA in Terms of CBI'

Substance Reference CPtb . ,iogenic Potencyb Aflatoxin 6, Garner and Wright (10) 17,000 Dimethylnitrosamine Grilli ct al (1 2) 2,310 Strong Aflatoxin MI Lutz (22) 1,600

2-Acetylaminofluorene Goodman ct a1 (1 1) 560 Vinyl chloride Bolt el al (5) 525 1,2-Dibrorn~ethane~ Arfellini et al (1) 515 Moderate o-Aminoazotoluene Lawson and Dzhioev (21) 230

1,2-Dichloroethane Arfellini et al (1) Carbon tetrachloride Rocchi et al (35) Urethan Prodi ct al (31) Epichlorohydrin' Mazzullo el al (24) Ethionine Crilli et al ( 1 3)

47 4Gd 34 Weak 23

1

a-Hcxachlorocyclohexane Sagelsdorff et al (37) co. 1 Cocarcinogen or promoter Saccharin' Lutz and Schlatter (23) <0.005

a Calcutated according to Lutz (22). Classification of CBI values with respect to oncogenic potency (22): in the thousands, strong; in the hundreds,

The hepatocarcinogenic effect of such oncogenic chemicals has not been proven up to now. Mouse liver instead of rat liver. ' A nonhepatocarcinogen able of inducing bladder tumors (CBI value in bladder DNA ~0.05).

moderate; in the tens, weak for initiators; below 1 for nongenotoxic oncogens (promotors and cocarcinogens).

tween extent of binding and concentration of tracer or microsomal protein further con- firms that an enzymatic pathway is involved in the binding process. The addition of cyto- solic fractions and/or GSH to NADPH-con- taining microsomal system strongly inhibits the extent of interaction with DNA. There- fore, an activation of the chemical by cyto- solic pathway, seldom found as in the case of 1,2-dibromoethane, does not occur. Such a finding confirms the protective role of gluta- thione from attack by electrophilic alkylating metabolites to nucleophilic sites and suggests that the CBI of bromobenzene in vivo could be underestimated by the effectiveness of GSH conjugation occurring in liver. Indeed, the covalent binding of the toxic metabolite of bromobenzene to liver proteins was com- pletely inhibited even at 0.1 M glutathione in vitro (41), whereas the covalent binding of bromobenzene to liver proteins in vivo was detected mainly when liver glutathione con- centration was less than 0.1 mM (171.. The strong inhibition of microsome-mediated binding by GSH suggests that not only the 3,4-oxide derivative of bromobenzene but also a phenolic metabolite is involved in the interaction process (14). Catechol-like diphe- nols, such as the 3,4-diphenol, may be further oxidized to semiquinones and quinones by superoxide radical anions (38). A participa- tion of this oxygen radical is thus suggested

by the effect of superoxide dismutase which inhibited the binding of bromobenzene by 50% (15). Recent studies suggest that the ox- idative stress determined by the generation of activated oxygen species and the subse- quent lipid peroxidation may offer an alter- native to covalent binding as an explanation of the biologic activity of many hepatotoxins. The interpretation of the role of lipid perox- idation in liver cell.injury by aryl halides could be more complex. It may be related to the depletion of GSH which renders the liver cell more susceptible to oxidative stress. Nev- ertheless, the covalent binding of electro- philic metabolites of hepatotoxins, such as bromobenzene, chlorobenzene, and iodo- benzene, to nucleophilic sites of macromole- cules is still considered to be the major mech- anism of hepatotoxicity (9).

The microsome-mediated interaction of bromobenzene to DNA in vitro resembles that of 1,2-dibromoethane, a halogenated al- kane recently studied by us under similar experimental conditions (8). Lung micro- somes from mouse are strongly active in ef- fecting the interaction with DNA of both bro- mobenzene and. 1,2-dibromoethane, whereas rat lung microsomes are inactive, and 1,2- dibromoethane induces lung tumors in mice but not in rats (28). The noticeable efficiency of hepatic microsomal system in bioactivat- ing bromobenzene in vitro is in agreement

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Vol. 13, No. 4, 1985 BROMOBENZENE BINDING TO DNA 281

with the pattern of in vivo binding. The com- parison between in vivo and in vitro inter- actions shows some differences. The labeling of mouse kidney DNA is high, while micro- somes from this organ are unable to mediate the in vitro interaction with DNA. They, however, catalyze the binding to microsomal RNA and proteins.

An opposite situation is seen with mouse lung since enzymatic fractions are very effi- cient in vifro, whereas the in vivo binding of bromobenzene with lung DNA is very low. It is probable that binding to kidney DNA in vivo is due to a reactive intermediate pro- duced by hepatic microsomes with a stability sufficiently high to allow “at distance” effects (26). In the case of mouse lung in vivo, the reactive forms locally produced by micro- somes and intermediates produced by liver metabolism would not be able to penetrate into the nucleus and to interact with DNA. An interference by cytosolic pathway and/or the presence of GSH could also explain such a discrepancy. In conclusion, the evidence presented here shows that bromobenzene in- duces covalent binding to nucleic acids in in vivo and in vifro systems, providing unques- tionable proof of its genotoxicity. Previous data from short-term assays were either mar- ginally positive in a polA- test in the absence of activating system (36). or negative in a morphologic transformation in the absence of metabolizing enzymes (29), and in a n Ames test using Aroclor as inducer of microsomes (25). Nevertheless, bromobenzene is not a direct alkylating agent and needs metabolic activation to show biologic effects. Also, the Aroclor induction of mixed function oxidase system resembles that of both PB and 3-meth- ylcholantrene and the effect of the last poly- nuclear aromatic hydrocarbon on bromoben- zene metabolism is quite opposite to that of PB. Therefore, the data now available suggest that long-term assays should be planned in order to detect the potential oncogenic action of bromobenzene since it is a halo derivate of benzene, a well-known carcinogen in hu- mans and in animals. Bromobenzene is more active than the parent compound to cova- lently interact with nucleic acids in biologic systems (27). There are already reports in the literature on chemicals which at first were found genotoxic in short-term tests and then oncogenic in long-term assays for carcinogen- icity. Finally, the photo-induced binding of bromobenzene mediated by X = 254 nm might involve the formation of free radicals

.

as intermediates, related to its high c value at such wavelength due to the presence of a benzene ring in the molecule. In fact, near- ultraviolet irradiation gives rise to a negligi- ble binding, although a 21-fold higher inci- dent fluence rate was used. Therefore, the environmental contaminant bromobenzene is activated to reactive forms by ultraviolet light like other carcinogens, such as dimeth- ylnitrosamine (3), and polycyclic aromatic hydrocarbons (30) regardless of the similari- ties of biologic and photochemical pathways involved in the interaction process which were confirmed (3) or not supported (30). Thus, a possible risk for humans could exist since covalent binding to DNA is a crucial event in the process of chemical oncogenesis.

ACKNOWLEDGMENTS SKI: 525-A was a kind gift from Smith,

Kline and French, Welwyn, UK. This work was supported by a grant from Minister0 della Saniti, Contract 500.4/RSC/135/L/2353, Rome, Italy.

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