Document O1nKeE89g16v2x49XjyovXJ61

.. ,-+NCER RESEARCH 49, 164-161, Jnnitary I , 1989) Reactivities of benzene metabolites (phenol, catechol, hydroquinone, I,4-benzoquinone, 1.2,4-benzenetriol) and related polyphenols (resorcinol, pyrogallol, phloroglucinol) with DNA were investigated by a I>NA sequencing technique using "P 5'-erid-labeled DNA fragnients obtained quences obtained from protooncogene (c-Ha-rus-I). 1,2,4-Benzerietriol was shown to cause strong DNA damage, and the parlicipation of active oxygen spccies was investigated. from hiininn c-l la-rus-l protooncogene, and the reaction niechanisni was studied by UV-visible and electron-spin resonance spectroscopies. 1,2,4- Ilenzcnctriol caused strong DNA damage even without alkali trcntmcnt. MAI`ERIALS AND ME`I'I IODS Alkali-labile sites induced by I ,2,4-benzenetriol were base residues of giinnine and adjacent thymine. Catalase. superoxide dismutase and mctliional inhihited the DNA damage coinpletely, but sodium formate did not inhibit it. 1,2,4-Denzenetriol-induced DNA damage was inhibited by the addition of a Cu(1)-specific chelating agent, bathocuproine, and was accelerated by the addition of Cu(l1). The addition of Fe(l1l) did not create any significant effects on 1,2,4-benzenetriol-induced DNA damage. Electron-spin resonance studies using spin traps demonstrated that addition of Fe(II1) increased hydroxyl radical production during the autoxidation of 1,2,4-benzenetriol, whereas the addition of Cu(I1) did not. The results suggest that DNA damage was caused by an unidentified active species which was produced by the autoxidation of 1,2,4-benzenetriol in the presence of Cu(Il), rather than by hydroxyl radicals. The possihility that I,2,4-benzeiietrioI-indiiced DNA damage is one of the primary reactions in carcinogenesis induced by benzene is discussed. Materials. Restriction cnzynics (B.siEII, Aeal, Xhal, f s r l ) and T, polynucleotide kinase were purchased from Toyobo Co., Osaka, Japan. Calf intestine phosphatase was obtained from Boehringer Mannheim GnibH. [y-"PjATP (6000 Ci/mmol) was supplied by New England Nuclear. Phenol, catechol, hydroquinone, I,4-benzoquinone, quinhy drone, resorcinol, pyrogallol, phloroglucinol, and sodium formate were purchased from Nakarai Chemicals Co., Kyoto, Japan. DTPA,) and bathocuproincdisulfonic acid were from Dojin Chemicals Co., Kuma. moto, Japan. Desferrioxamine mesylate was from Ciba-Geigy. Agarose was from Takara Shuzo Co., Kyoto, Japan. Acrylamide, bisacrylamide, and piperidine were from Wako Chemicals Co., Osaka, Japan. Hydra. zine was from Eastman Organic Chemicals. SOD (3100 unit/mg from bovine blood), catalase (45,000 unit/nig from bovine liver) and metliional were from Sigma Chemical Co. 1,2,4-Benzenetriol, DMPO, and dimethyl sulfate were purchased from Aldrich Chemical Co. A INTRODUCTION stock solution of 0.1 M polyphenol in acetone was kept at O'C in the dark and used within 4 h of preparation. Benzene, widely used in the chemical industry, has been shown to cause serious hematological disorders and carcinogenic effects on humans and animals. Extensive epidemiological evidence has been published on the high incidence of leukemias Preparation of 32PS'-End-labeled DNA Fragments. DNA fragments were prepared from plasmid pbcNI purchased from American Type Culture Collection, which carries a 6.6 kilobase BamHl chromosomal DNA segment containing human e-Ha-rus-1 protooncogene ( I 5, 16). The plasmid was digested with BstElI and A w l , and tlie resulting DNA in men occupationally exposed to benzene (1, 2). The admin- fragments were fractionated by electrophoresis o n 1 or 2% agarose gels. istration of benzene to animals has produced leukemia ( 3 ) , lymphoma and carcinomas of the Zymbal gland, maniinery gland and liver ( I , 4). Previous studies have demonstrated that benzene induced sister chromatid exchanges in mouse bone marrow ( I , 5, 6). However, benzene has not been shown to be mutagenic in bacterial test systems. It is well accepted that many carcinogens are readily converted to reactive intermediates by drug metabolizing enzymes. A variety of DNA-carcinogen adducts and oxidized bases have A "P S'-end-Iabeled 351 base pairs Aval fragment ( A w l * 1294-Avol' 1644) from 1.5 kilobase BstEll fragment of pbcN1 was obtained by dephosphorylating with calf intestine phosphatase and repliosphorylating with [y-"PJATP and T, polynucleotide kinase. The singly labeled 261 base pair fragment (Aval* 1645-XbaI 1905) and 341 base pair fragment (Xbol 1906-AeaI* 2246) were obtained by digesting the '*P 5`-end-labeled 602 base pair Avo1 fragment (AvaI* 1645-Awl* 2246) from 1.5 kilobase BsfEIl fragment with Xbal. The singly labeled 98 base pair fragment (AvaI* 2247-Psd 2344) was obtained by digesting the '*P 5'-end-labeled 435 base pair Avo1 fragment (Aval* 2247-Aiwl' been identified in D N A recovered from reactions with carcin- 2681) with PsfI. The asterisk indicates 32P-labeling and nucleotide ogens (7). However, in the case of benzene, little evidence for direct damage to DNA has been presented with the exception of evidence for covalent binding of labeled benzene with DNA (8,9). Furthermore, the ultimate carcinogenic forms of benzene and the mechanisms of their reactions with DNA remain to be clarified. I k n z c n e mckibolisrii is considcrcd to bc ililportilnt for tlic expression of its toxicity (10). Benzene metabolites include phenol, catechol, hydroquinone and 1,2,4-benzenetriol(lO-14). numbering starts with the BomHI site (16). Detection of DNA Damage Induced by Benzene Metabolites. The standard reaction mixture in a microtube (1.5-ml Eppendorf) contained 2.5 mM o r 5 mM benzene and its metabolites and ["PIDNA fragmenl (-5000cpm) in 200 pl of 20 mM sodium phosphate buffer (pH 7.9) or Tris-HC1 buffer (pH 8.0). After incubation for 30 min or 9 0 min at 37'C.tlie DNA fragments were precipitated with cold ethanol and dried in ii v:icuuni dcsicc:itor, followcd by hcnting :it YO'C for 25 niin in I M piperidine when necessary. The DNA fragments were electrophoresed and the autoradiograms were obtained as previously described (17). Resorcinol, pyrogallol ( 1,2,3-benzenetriol) and phloroglucinol The preferred cleavage sites by 1,2,4-henzenetriol were determined by (1,3,5-benzenetriol) have not been identified as benzene metab- direct comparison of tlie positions of the oligonucleotides with those produced by the chemical reactions of the Maxam-Gilbert procedure Received 5/5/88: revised 9/2/88; accepted 9/30/88. The costs of publication of this article were defrayed in part by the payment of page cliarps. This article must thcreforc be licrchy innrkcd n~lrertircnrenfin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'This work was supported in part by a research grant from the Fujiwara Foundation of Kyoto University and Grant-in-Aid lor Scientific Research (62570228 and 63870027) from the Ministry of Education, Science and Culture of Japan. To whom requestcfor reprints should be addressed. (18). A laser densitometer (LKB 2222 UltroScan XL) was used for the nic:Isiircnicnt of thc relative amounts of oligoniiclcotidcs produced by the incubation of DNA fragment with I ,2,4-benzenctriol. 'The abbreviations used are: DTPA, diethylenetriamine-N'JV"JV"-pen- taacetic acid; SOD, superoxide dismutase; DMPO, 5,5-dimethylpyrroline-Noxide: DMPO-OH. hydroxyl radical adduct of 5.5-dimethylpyrroline-~-oxide: ESR, electron-spin resonance. I64 DNA DAMAGE BY I,2.J-BENZENECHIOL UV-visible Spectra bleasurements. UV-visible spectra were measured triol >> hydroquinone > phloroglucinol, pyrogallol, catechol, 41 37'C with a UV-Vis-NIR recording spectrophotometer (Shi- resorcinol, phenol and benzene. Benzoquinone and quinhy- ni;ldztr UV-365). drone (an addition compound of one mol hydroquinone and ESR Spectra Measurements. ESR spectra were measured at room one mol 1,4-benzoquinone) caused little or no damage to D N A leniperature using a JES-FE-3XG spectronieter with 100-KHz field (data not shown). ;e- modulation according to the method previously described (17, 19). Spcciri were recorded with a microwave power of 4 m W and a modulation amplitude of I .O G . T h e magnetic fields were calculated by the splitting of Mn2' in MgO (At()-, = 86.9 G ) . DMPO was used :IS a spin Effects of Scavengers, Chelating Agents, and Metal Ions on 1,2,J-Benzenetriol-induced DNA Damage. Fig. 2 shows the effects of SOD, catalase, and hydroxyl radical scavengers on :n- trapping reagent. T h e measurements were carried out after the incu- 1,2,4-benzenetriol-induced DNA damage. S O D and catalase he bation of 1,2,4-benzenetriol in a buffer solution. CuCll or FeCI, solution almost completely inhibited D N A damage (Fig. 2, Lanes 3 and was added where indicated. 4), suggesting the involvement of superoxide and hydrogen peroxide. The addition of hydrogen peroxide enhanced DNA RESULTS damage to a great extent (data not shown). Methional significantly inhibited DNA damage (Fig. 2, Lane 6).whereas sodium Damage of 32P-labeled DNA Fragments Induced by Benzene formate did not inhibit it (Fig. 2, Lane 5 ) . T4 hletabolites and Related Polyphenols. The extent of DNA dam- Since buffers and reagents are known to be invariably contam- . in. age was estimated by gel electrophoretic analysis. Fig. 1 shows inated with trace amounts of metal ions (20), and copper ion is iin nd an autoradiogram of DNA fragments treated with bcnzcne and reported to accelerate drug-induced DNA damage (2 I), the its metabolites. The upper band and lower band in the control effects of chelating agents and metal ions on 1,2,4-benzenetriol- iY:re show double-stranded and single-stranded forms of DNA frag- induced DNA damage was examined. 1,2,4-Benzenetriol-in- iid nient, respectively. In the case of I,2,4-benzenetriol, oligonu- duced DNA damage was inhibited by the addition of a &(I)- la- cleotides were clearly detected on the autoradiogram as a result specific chelating agent, bathocuproine (Fig. 3, Lane 3), and Ise 01' DNA cleavage even without alkali treatment (Fig. 1, Lane was accelerated by the addition of Cu(l1) ion (Fig. 3, Lane 6). le, 5), suggesting breakages in the deoxyribose-phosphate back- On the other hand, a Fe(ll1)-specific chelating agent, desfer- 'a- bone by active species. -i'tie amount of oligonuckotidcs in- rioxariiine did not inhibit D N A damage significantly (Fig. 3, m creased with alkali treatment, suggesting that the base altera- Lane 4, and Fe(ll1) did not accelerate it (Fig. 3, Lane 7). ie- tions were induced by 1,2,4-benzenetriol. The DNA cleavage DTPA inhibited DNA damage (Fig. 3, Lane 5 ) , suggesting that 0, A he by I,2,4-benzenetriol increased with time (data not shown). DTPA functions as a copper-chelating agent. DNA cleavage induced by hydroquinone was much less than Alkali-labile Sites of DNA Fragments Treated with 1,2,4- that induced by I,2,4-benzenetriol (Fig. 1, Lane 2), and no Benzenetriol. To estimate the site specificity of DNA cleavage its cleavage was observed with benzene, phenol, catechol or resor- by 1,2,4-benzenetriol, 32P 5'-end-labeled DNA fragments pe cinol even with alkali treatment. treated with 1,2,4-benzenetriol were electrophoresed and the al Little or no cleavage was observed with pyrogallol or phlo- autoradiogram was scanned with a laser densitometer (Fig. 4). i). roglucinol even with alkali treatment (data not shown). The The cleavage sites induced by I ,2,4-benzenetriol were deter- IA relative reactivities with DNA were as follows: I ,2,4-benzene- mined by utilizing the Maxam-Gilbert procedure (17). The Is. result showed that cleavages at the positions of guanine and I* 'Y It:d 1234567 adjacent thymine were more frequent than those of other bases. The cleavages occurred broadly at or near the 12th codon of cHa-rus-1 protooncogene under the present conditions (Fig. 4). ir 'P 5) 18 123456 'g I* le le d It )r It d 4 d 1. Y e e r Fig. 2. Effect of SOD, catalase and hydroxyl radical scavengers on DNA Y Fig. I . Autoradiogram of "P-labeled DNA fragments incubated with benzene damage induced by 1,2,4-benzenetriol. The '%P 5'-end-labeled 341 base pair metabolites.Reaction mixture contained "P-labeled 35 I base pair DNA fragment fragment (Xbal 1906-Aval* 2246) in 200 pl of 20 mM phosphate buffer at p H (.lvul* 1294-Aval' 1644) and 5 nihi benzene or its metabolites in 200 pl of 20 7.9 was incubated with 2.5 mki 1,2.4-benzene1riol (Lane 2). I.2,4-benzenetriol nici Tris-HCI buffer at pH 8.0. Lane I , control; Lane 2, hydroquinone; Lune 3, plus 30 units of SOD (Lane 3). 1.2,4-benzcne1riolplus 30 units ofcatalase ( h i e cltechol; Lane 4. resorcinol; Lane 5, I.2,4-benzenetriol; Lane 6, benzene; Lane 7, 4). i,2,4-benzenetriol plus 0.1 M sodium formate (Lune 5). and 1,2.4-benzenetriol plienol. After incubation b r 9 0 iiiiii at 3 7 T , cold ethanol was addcd. l l i c plus 0.1 hi niethioiial (Lane 6) for 30 inin at 37'C. Lane I shows the electropho- precipitated UNA fragments were electrophoresed on an 8%polyacrylamide/8 hl resis pattern of untreated "P-DNA fragment. The treated DNA fragments were urea gel and the autoradiograin was obtained by exposing X-ray film IO the gel. analyzed by the nielliod described in the legend to Fig. I . 165 DNA DAMAGE BY 1.2.4-RENZENEI'RIOL 1234567 12 A 11 I 10 Lr) 30 09 8 mv) O 8 Q 07 06 Fig. 3. Effect of chelating agents and metal ions on DNA damage induced by 1,2,4-benzenetriol. The '*P 5'-end-labeled 261 base pair fragment ( A w l * 1645Xbul 1905) in 200 pl of 20 mM phosphate buffer at pH 7.9 was incubated with 2.5 mM 1,2,4-benzenetriol (Lone2), 1,2,4-benzenelriol plus 5 p M bathocuproine (Lone 3), 1,2,4-benzenetriol plus 5 p~ desferrioxamine (Lune 4), 1,2.4-benzenelriol plus 5 pM DTPA (Lone 5). 1.2,4-benzenetriol plus 10 pM CiiC12( I m e 6). and 1.2.4-1~'1l~.eiietripoliis I O p~ I~e('l.,( / m w7) for 30 inill 111 37.C:. Irtrtc, 1, electrophoresis pattern of untreated I'*P]DNA fragment. The treated DNA fragments were analyzed by the method described in Fig. I. legend. UV-Visible Spectroscopic Studies on the Autoxidation of 1,2,4-Benzenetriol. Fig. 5 shows changes in the UV-visible spectra of 1,2,4-benzenetriol with time. When a stock solution of 1,2,4-benzenetriol was added to a buffer solution, the solution turned immediately red with an absorption maxima at 482 nm and 267 nm. These absorption maxima can be attributed to 2hydroxybenzoquinone (22). The conversion from 1,2,4-benzenetriol to 2-hydroxybenzoquinone was inhibited to over 95% by SOD, but was not affected by the addition of Fe(II1) or Cu(I1) (data not shown). The intensity of absorption at 482 nm began to decrease within about several seconds. Autoxidation of pyrogallol and hydroquinone was slower and simpler than that of 1,2,4-benzenetriol. Under the same conditions, phenol, resorcinol, catechol, and phloroglucinol showed little spontaneous autoxidation. F'roduction of Free Radicals during the Autoxidation of 1,2,4Benzenetriol. Spin trapping methods were used to detect free radicals produced during the autoxidation of 1,2,4-benzenetriol. Fig. 6A, shows an ESR spectrum of a spin adduct observed when D M P O was added to a buffer solution o f 1,2,4-benzenetriol after incubation a t 37C for 15 min. The 1:2:2:1 pattern of four lines can be interpreted in terms of equivalent hyperfine splitting constants (UN, (I") due to both the nitroxide nitrogen atom and the @-hydrogen.The spin adduct (UN = uH = 14.8 G) can be reasonably assigned to DMPO- OH by reference to the reported constants (17, 19, 23, 24). Catalase completely inhibited the yield of D M P O - O H (Fig. 6B). Fe(1II) increased the yield of DMPO-OH (Fig. 6C).O n the other hand, when Cu(1l) was added, the yield of D M P O - O H did not increase, but a sixline signal with equal intensities (aN = 15.7 G, uH = 22.9 G) could be detected (Fig. 6 0 ) . The six-line signal was observed with Cu(1I) alone. DISCUSSION The present results showed that among benzene metabolites, 1.2,4-benzenetriol caused strong DNA damage, and hydroquinone caused slight DNA damage. Benzene, phenol, catechol, and resorcinol showed no effect. Trace amounts of copper ion were shown to be necessary for the induction of DNA damage by 1,2,4-benzenetriol. 05 I 1 680 12 B 11 8IO z 2 d OS 2 <m 08 1690 1700 I 1710 II 1720 173 07 0.6 !5 I 2220 I8 C 16 !a ez " 2Q I2 SI m 4 lo I 2210 I 2200 I 2190 I 2180 2265 I 1270 --L----l_____ 2280 2290 2300 NUCLEOTIDE NUMBER 2310 Fig. 4. Alkali-labile sites in "P-labeled DNA fragments incubated with 1,Z.J. benzenetriol. A, "P S'-end-labeled 261 base pair fragment (Avul' 1645-Xbrl 1905) in 200 pl of 20 mM phosphate burner at pH 7.9 was incubated with 2.5 mu 1,2,4-benzenetriol lor 30 min at 37'C. E, P' 5'-end-Inbeled 341 base prb fragment (Xbul 190dAml* 2246) was used. C,'*P 5'-end-labeled 98 base pa# fragment (Avul' 2247-Psfl2344) was used. After the piperidine treatment, DNA fragments were electrophoresed on an 8% polyacrylamide/S M urea gel and Ik autoradiogram was obtained by exposing X-ray film to the gel. The relalilt amounts of oligonucleotides produced by incubation with 1,2,4-benzenetriol w ~ n measured by a laser densitometer (LKB 2222 UltroScan XL). The alkali-labk sites of DNA induced by 1,2.4-benzenetriol were determined by direct cornparim with the same DNA fragment after undergoing the DNA sequence rearlion according 10 the Maxam-Gilbert procedure ( I 8). Horizonlo/ a i s , nucleolidc number of human c-Ha-rm-1 prolooncogene starting with the EamHI site (151 A, G, T,and C,deoxyadenylate. deoxyguanylate, deoxythymidylale, and dcor) cytidylate of DNA, respectively. Underscoring, the I21h codon of human c.ll8 rus-1 protooncogene. experiments using DMPO detected the production of hydroxyl radicals after the incubation of 1,2,4-benzenetriol in a buffer solution. Cu(I1) did not show any effect on the production of hydroxyl radicals, although the addition of Cu(1I) accelerated the DNA damage dramatically. Fe(II1) accelerated the produc- tion of hydroxyl radicals whereas Fe(1II) did not show any accelerating effect on 1,2,4-benzenetrioI-inducedD N A damage. In addition, the yield of hydroxyl radical seems to be much less than the degree of 1,2,4-benzenetriol-induced D N A damage. Methional completely inhibited 1,2,4-benzenetriol-induced DNA damage, whereas sodium formale, anotlicr hydroxyl rad- ical scavenger, did not inhibit it. Although methional is a W A Y E L E NGTH(nm) Fig. 5. Changes in UV-visible spectra of a solution of 1,2,4-benzenetriol with time. Visible region (400-600 nm) was recorded using 1-cm quartz cuvets and UV region (200-400 nm) was done using 1-mm quartz cuvets at 37'C. To 3.92ml phosphate buffer (20 mM, pH 7.9), 80 pl of a stock solution of 0.1 hl 1,2,4knzenetriol in acetone was added. Spectral tracing was recorded every 10 min. hydroxyl radical scavenger, it can also scavenge other radicals (26). Therefore, it is considered that the species causing DNA damage is an unidentified active species rather than hydroxyl radical. The inhibitory effect of catalase and the increasing effect of hydrogen peroxide on D N A damage suggest that hydrogen peroxide participates in the production of active species. The inhibitory effect of bathocuproine and the accel- erating effect of Cu(l1) o n D N A damage suggest that an inter- conversion of Cu(I1) and Cu(1) has a n important role in the production of active species. The DNA sequencing experiments on DNA fragments B treated with 1,2,4-benzenetriol revealed that cleavages at the positions of guanine and thymine were more dominant than those of adenine and cytosine. Cleavages of guanine and thy- mine residues in the guanine-rich region were especially pre- dominant. Our previous paper suggested that hydroxyl radicals caused cleavages at every nucleotide with slightly stronger cleav- ages occurring at every guanine and thymine position (17). This difference may support the idea that D N A damage induced by 1,2,4-benzenetriol was caused by unidentified active species rather than hydroxyl radicals. Recently it has been demon- strated that mutations activating human c-Ha-rus-1 protoon- D cogene are induced by chemical carcinogens (27). This preliminary experiment suggests that active species produced during the autoxidation of 1,2,4-benzenetriol broadly attacked nucle- - otides of the 12th codon and its neighboring regions. T h e idea that damage to D N A is a critical event in the initiation of carcinogenesis is generally accepted. Recent obser- 10 G Fig. 6. ESR spectra of the hydroxyl radical spin adduct of DMPO produced during the autoxidation of 1,2,4-benzenetriol. A, sample (100 p l ) contained 2.5 mM I,2,4-benzenetriol in 20 m M phosphate buffer at pH 7.9; B, catalase (24 units) was added to the sample solution; C,FeCl, (10 p ~ w)as added to the sample solution; D, CuCI, (2 mM) was added to the sample solution. After incubation for 15 min at 37.C. 0.3 M DMPO was added, aliquots of the solution were taken up in a calibrated capillary, and ESR spectra were measured at room temperature as described in "Materials and Methods." vations have suggested that some tumor promotors act to produce DNA damage via free radicals (28,29). The importance of this process in the carcinogenesis of benzene remains to be clarified. The present study shows that 1,2,4-benzenetrioI is autoxidized in the presence of copper ion to produce an active species causing DNA damage and may play an important role in benzene-induced carcinogenesis, although the role of hydroquinone cannot be excluded. After benzene exposure, catechol UV-visible spectroscopic studies suggest that 1,2,4-benzenetriol is autoxidized rapidly in aqueous solution. T h e initial increase in the absorption maximum at 482 nm seems to correspond with the autoxidation of I ,2,4-benzenetriol to form 2-hydroxybenzoquinone and hydrogen peroxide through the intermediates of semiquinone and superoxide anion. The inhibitory effect of S O D on autoxidation, observed in this and previous studies (25), suggests that the superoxide anion participates as an initiation and chain propagating species during the first step in the autoxidation of 1,2,4-benzenetriol. Cu(l1) and Fe(II1) did not affect the initial autoxidation. In an attempt to interpret llie mechanism of DNA damage induced by 1,2,4-benzenetriol, we examined what kind of active species are produced and cause D N A damage. Spin trapping and hydroquinone were shown to be retained in the bone marrow of rats in higher concentrations and for longer periods than phenol (30). I t is possible to speculate that catechol is converted to 1,2,4-benzenetrioI, resulting in DNA damage. Recently, Pellack-Walker and Blumer have reported that 1,4benzoquinone may cause DNA damage in cells more efficiently than 1,2,4-benzenetriol (31). In the present results, 1,4-benzoquinone itself did not cause DNA damage. This discrepancy may be explained by the difference in permeability or stability between 1,2,4-benzenetriol and 1,4-benzoquinone, or by the possible activation of 1,4-benzoquinone in cells (32). Further research is necessary to clarify whether or not I ,2,4-benzenetriol induces DNA damage B vivo as a critical event in the initiation and/or promotion of carcinogenesis by benzene. 167 - .i DNA DAMAGE BY 1.2.4-BENZENETRIOL ITA ACK NO\\:l .EDGR I EN'I'S \\'e are grntefitl to Professor Seiyo Sano f o r his encoitrageiiient tlirougliout this work. REFERENCES I . IARC Working Group. Sonie Industrial chemicals and dyestuffs. In: I A R C Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Vol. 29, pp. 93-148. 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