[CANCER RESEARCH 46, 1654-1658, April 1986]

Genotoxicity of a Variety of Azobenzene and Aminoazobenzene Compounds in the

Hepatocyte/DNA Repair Test and the SVï//n0/ie//fl/MutagenicityTestHideki Mori,1 Yukio Mori, Shigeyuki Sugie, Naoki Yoshimi, Masayoshi Takahashi, Hiroaki Ni-i, Hiroshi Yamazaki,

Kazumi Toyoshi, and Gary M. WilliamsDepartment of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500, Japan [H. M., S. S., N. Y., M. TJ; Laboratory of Radiochemistry, GifuPharmaceutical University, 6-1, Mitaborahigashi, Gifu 502, Japan [Y. M., H. N., H. Y., K. TJ; and Naylor Dana Institute, American Health Foundation, Valhalla, NewYork ¡0595¡G.M. W.]

ABSTRACT

Genotoxicity of 39 azo dye compounds of azobenzenes, aminoazobenzenes, and diaminoazobenzenes was examined in the hepatocyte primaryculture/DNA repair test. Azobenzene (AzB) and 3,3'- or 4,4'-substituted

azobenzenes such as (( 11,): \/H. «l M >ll ), w»,(CH2OCOCHj)jAzB,and (( 11:( lh \ / H did not generate DNA repair, indicating lack of geno-toxicity of these compounds. In contrast, all of 24 aminoazobenzenes,including those of unknown carcinogenicity, i.e., 3'-methyl-4-aminoazo-benzene, 3'-CHiOH-aminoazobenzene, 3'-hydroxymethyl-W-methyl-4-aminoazobenzene, 3'-COOH-methylaminoazobenzene, 4'-fnrmyI-.v..V-dimethyl-4-aminoazobenzene, 3'-CHiCl-dimethylaminoazobenzene, 4'-CH2CI-dimethylaminoazobenzene, and 2'-, 3'-, or 4'-( II2(>( (M 11,-di-

methylaminoazobenzene, elicited DNA repair synthesis. A positive DNArepair response was obtained for the 3 of 6 tested diaminoazobenzenes,i.e., yV'-acetylWV'-methyM-amino-dimethylaininoazobenzene, V-acc-tyl-A/'-methyl-4-amino-methylaminoazobenzene, and .V'-:uvt>l-.V-

methyl-4-amino-yV-acetyl-methylaminoazobenzene, which are known tobe carcinogenic. These results indicate that the amino group is functionalfor the expression of genotoxicity of azobenzene compounds. Twenty-oneazobenzenes of these 3 classes were also examined for their mutagenicityin the 5a/m0ne//a/mutagenicity assay. These results were almost identicalwith those of the DNA repair test except for several azo dyes such asAzB and 4.4'-« I l;Oannl); \/R of the azobenzenes and V-ai-i-tyl-4-amino-dimethylaminoazobenzene and Ar/-acetyl-Ar-methyl-4-amino-yV-

acetyl methylaminoazobenzene of the diaminoazobenzenes.

INTRODUCTION

A wide variety of azo dyes including carcinogens has beenidentified (1-3). Among these, aminoazo dyes have been studiedextensively (2,4-9). Some carcinogenic aminoazobenzenes substituted with a methyl group, such as 3'-CH3-DAB2 or 3'-CH3-

MAB, are more carcinogenic than their parent compounds (2),whereas other substituted aminoazobenzenes such as 2'-('H <-DAB or 4'-CH3-DAB are less carcinogenic (5). The mecha

nisms of the differential carcinogenicity of these agents are notwell understood. Previously, Mori et al. (10-15) documentedthat metabolic oxidation occurs at the ring methyl group of 3'-CHj-DAB (10) or 3'-CH3-MAB (16) to yield the 3'-CH2OH,3'-CHO, and 3'-COOH derivatives and found potent mutagenicity (11, 17) and carcinogenicity (12, 14, 15) of 3'-CH2OH-DAB and its W-demethylated derivatives. These findings suggest that metabolic oxidation plays an important role in thecarcinogenesis of 3'-CH3-DAB and 3'-CH3-MAB. Samuels etal. (18) also reported that the oxidative metabolites of 3'-CH3-

DAB were detected in the liver and bile of rats.Various azo compounds structurally related to the com

pounds with the ring methyl group and other symmetrically

Received 5/17/85; revised 9/18/85, 12/11/85; accepted 12/26/85.1To whom requests for reprints should be addressed.2The abbreviations used are: DAB, JV./V-diinethyl-4-ammoazobenzene; MM».

A-methyl-4-aminoazobenzene; AzB, azobenzene; AB, 4-aminoazobenzene;DADAB, W ilimciliU 4' aminn V\ clum-ilul 4 .iimmi.i/obcii/LMK-:MA, JV'-methyl-4-amino: MADAB, A''-methyl-4'-amino-A'^V-dimethyl-4-aniinoazoben-zene; ADAB, 4'-amino-AyV-dimethyl-4-aminoazobenzene; M AMAB, W-methyl-4'-amino-Ar-methyl-4-aminoazobenzene; Ac, acetyl; HPC, hepatocyte primary

culture; UDS, unscheduled DNA synthesis; S9, 9000 x g supernatant.

substituted azo compounds or their acetyl- or chloro-derivativeshave been prepared (10, 11, 13,19), but it is impractical to testall of these for carcinogenicity in vivo. In vitro short-term testsoffer an attractive alternative. Since the carcinogenicity of azo-benzene-type compounds has been manifested mainly in theliver (1,2, 8), the ideal test system for these compounds wouldbe one derived from liver.

The HPC/DNA repair test developed by Williams (20, 21)has been shown to respond to carcinogens of a variety ofstructural classes (22-25), including azo dyes (21, 26). In thisstudy, 39 azo dye compounds representative of the azobenzene,aminoazobenzene, and diaminoazobenzene classes were studiedin the HPC/DNA repair test using rat hepatocytes. In addition,the Ames bacterial mutagenicity assay was also performed on21 azo dyes for which mutagenicity had not yet been studied.

MATERIALS AND METHODSChemicals. AzB, 3,2'-(CH3)2-AB, and 2'-COOH-DAB (methyl red)

were purchased from Wako Pure Chemicals Ltd., Tokyo, Japan. 4,4'-

Diaminoazobenzene derivatives were prepared according to the methoddescribed by Lin (27,28). All other aminoazobenzenes were synthesizedby Mori et al. of our group as described previously (10, 11, 13, 19).The synthesized products were identified by the measurement of melting point, elemental analysis, and spectroscopic determination (infrared, UV, and mass spectrometries). Prior to assay for the HPC/DNA repair or the mutagenicity, purity of the azo compounds wasdetermined to be more than 99% by high-pressure liquid chromatog-raphy. Structural properties of the azo dye compounds used in thisstudy are shown in Fig. 1.

HPC/DNA Repair Test. The procedure was basically that of Williams(21, 25) and Williams et al. (26) as described in a previous paper (22).Hepatocytes were isolated from the livers of male ACI rats weighing200 to 250 g. The isolated hepatocyes were allowed to attach on plasticcoverslips for 2 h in primary culture using Williams medium E. Thecultures were exposed to the test compounds and |MKV/M-/-'II |ihy midinc

(10 ¿iCi/ml,49 Ci/mmol; Amersham, United Kingdom) for 20 h. Allcompounds were dissolved in dimethyl sulfoxide in logarithmic dilutions before addition to the cultures. As positive control, A'-2-fluore-nylacetamide was used in the assays. The slides were dipped in SakuraNR-M2 photographic emulsion and exposed for 14 days. Autoradi-ographic grains were counted in a television (Olympus, type S) with amicroscopic attachment. The data were expressed as the average netcounts for 3 coverslips ±the SD (50 cells/coverslip). The test compounds were considered positive when the mean net nuclear grain countwas more than 5 grains above background and statistically greater thanthat of the controls. Two different assays were performed on every testcompound.

Sa/moneHa/Microsoine Test. Each test was carried out according tothe standard method of Marónand Ames (29) as modified by Mori etal. (11). Tester strains used were Salmonella typhimurium TA 100 andTA98. The liver S9 was obtained from male Sprague-Dawley rats (bodyweight, 180 g) pretreated with polychlorinated biphenyl mixture. Azocompounds (0.05-1.0 itmol) were dissolved in 0.1 ml of dimethylsulfoxide, except for DADAB (dimethyl formamide) and N'-AcMA-N-

AcMAB (methanol), and preincubated with or without the S9 mixtureat 37°Cfor 1 min (11, 12). The data were expressed as the average of

3 replicates.1654

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GENOTOXICITY OF AZO DYE COMPOUNDS

Diaminoazo benzenes

H, CHj, CH2OH, CHO, COW, C«2OAc or CHjCl

ABmDABR2HCH,CH,Azobenzenes

(AzB)R

\»5HHCH,DADABN'-AChADABN'-AcADABMMIr-AcWMBN'-ACHA-N-ACWB«1CH,»3H»3CM,fflj«2CH,COCH;COCH,HCOCH,COCH,«3CH,CH,CH,"3CH,CH,•«CH,CH,CH,HHCOCHj

« - H, CHj, CH2OH, CHjOAc Or CHjCI

Fig. I. Chemical structures of azobenzene derivatives.

RESULTS

Results of the HPC/DNA repair test are shown in Table 1.In the test, almost the same results were obtained from 2experiments, and therefore only the data from one experimentare shown in Table 1. As indicated in the table, the highest dose(10~4 M) of each compound was, in general, toxic for the

cultured cells. All 9 azobenzene compounds did not elicit DNArepair synthesis. In contrast, all 24 of the aminoazobenzenesproduced a positive response. Of these compounds, severalchemicals, i.e., 4'-CHO-DAB, 2'-COOH-DAB, 3'-CH2Cl-DAB, and 4'-CH2Cl-DAB, showed a lower UDS grain level

compared to the other aminoazobenzenes. Among the diami-noazobenzenes, N'-AcMADAB, N'-AcMAMAB, and N'-

AcMA-N-AcMAB generated UDS, but the other 3, i.e.,DADAB, N-AcADAB, and MAMAB, did not. Among the azodyes tested in the assay, positive chemicals exhibited a doseresponse in the UDS level.

Fig. 2 shows the results of the mutagenicity of AzB and 4,4'-

(CH2OAc)2AzB in the strain TA 100 of S. typhimurium with orwithout liver S9. Mutagenicity was clear with AzB and 4,4'-

(CH2OAc)2AzB in the presence and absence of liver S9, respectively. Both chemicals did not show mutagenicity on TA98 ineither the absence or presence of S9. All other azobenzeneswere negative for mutagenicity in both TA 100 and TA98 withor without S9 (data not shown). Fig. 3 shows the results on themutagenicity test of 6 aminoazobenzenes in TA 100 and TA98.2'-CH2OAc-DAB, 4'-CH2OAc-DAB, 3'-CH2Cl-DAB, 3,2'-(CH3)2AB, 2'-COOH-DAB, and 4'-CHO-DAB all demonstra

ted a positive reaction for mutagenesis in both strains withactivation by S9. However, 2'-COOH-DAB was negative in

TA 100. Fig. 4 indicates the results of the mutagenicity ofdiaminoazobenzenes in both strains of TA100 and TA98. Thesechemicals, N'-AcMADAB, N'-AcADAB, and N'-AcMAMAB,were positive in both strains with S9, except N'-AcADAB in

TA 100, but the other 3 diaminoazobenzenes were negative inboth strains with or without S9.

Table 2 summarizes a correlation between the results of theDNA repair test and the bacterial mutagenicity test and carcin-ogenicity of the tested azo dyes. Twelve aminoazobenzeneswhich have been reported as carcinogenic had positive reactionsin both assays of DNA repair and bacterial mutagenicity. Twoof the chemicals, 4'-CH2OH-DAB and 3'-CHO-DAB, for

which carcinogenicity is not yet clear, also showed positiveresponses in both tests. The results of the mutagenicity assayof two chemicals, AzB and 4,4'-(CH2OAc)2AzB, were in disa

greement with those of the DNA repair test. In the DNA repairtest, genotoxicity of the diaminoazobenzenes was consistentwith their carcinogenicities. Inconsistency between mutagenicity results and UDS or carcinogenicity findings was evidentwith two diaminoazobenzene derivatives, N'-AcADAB and N'-AcMA-N-AcMAB.

DISCUSSION

This study has delineated the genotoxicity of a variety ofazobenzene compounds, including simple azobenzenes, aminoazobenzenes, and diaminoazobenzenes.

None of the simple azobenzenes elicited DNA repair synthesis, and only two were mutagenic. Ring substituted azobenzenesare not known to be carcinogenic and, in fact, like aromaticamines, activation is believed to occur on the amino group (20).The reason for the mutagenicity of AzB and 4,4'-

(CH2OAc)2AzB is not yet clear. Since both chemicals showedmutagenicity only on strain TA 100, the mechanism of mutagenic activation might be different from that of aminoazobenzenes which were almost mutagenic in both strains. The mutagenicity of 4,4'-(CH2OAc)2AzB was apparent without liver S9,

and the chemical is ostensibly a direct-acting mutagen. Themutagenicity of the chemical, however, was not present withsupplement of S9. This may be related to possible inactivationby reaction with proteins and other substances in S9 and/orfurther metabolic inactivation such as azo-reduction, sincemany azo-reduced compounds are not mutagenic (11). Furthermore, the lack of DNA repair by this chemical might be relatedto the metabolic inactivation in the hepatocytes. It is of interestthat arylmethyl acetyl ester substituted azobenzenes showed adirect-acting mutagenicity. Recently, Watabe et al. (30, 31)demonstrated that arylmethyl sulfate esters of carcinogenicpolycyclic hydrocarbons are direct-acting mutagenic metabolites and react with rat liver cytosolic protein and DNA.Chloromethylbenzo(a)py-renes are also known to be direct mutagens in the Ames assay(32). No evidence, however, has been obtained for the metabolicactivation at the arylmethyl group in other carcinogens. Thus,the present result is the first evidence that a hydroxymethylester of azobenzene compound has a direct-acting mutagenicity,implying that 4,4'-(CH2OAc)2AzB is active per se or that the

bacteria are able to further metabolically activate the ester tomutagen. However, all other compounds having the same orsimilar functional groups, 3,3'-(CH2OH)2AzB, (CH2OAc)-

DAB, (CH2Cl)2AzB, and CH2C1-DAB, did not show the direct-acting mutagenicity, and 3'- or 4'-CHO-DAB, which could

potentially react with DNA directly through Schiff base formation, required the presence of S9 for mutagenicity. Accordingly, this suggests that bacterial DNA bases might not reactwith 4,4'-(CH2OAc)2AzB through a simple electrophilic reac

tion mechanism.In contrast to the findings with azobenzenes, all aminoazo

benzenes elicited DNA repair synthesis, indicating their geno-toxicities, confirming the findings of Williams (26). The resultswere almost identical with those for mutagenicity and correlatedwell with the carcinogenicity of these chemicals. Depending onthe reliability of the test system for the screening of carcinogenicity, several positive compounds, i.e., 2'-CH2OAc-DAB, 3'-CH2OAc-DAB, 4'-CH2OAc-DAB, 4'-CHO-DAB, 3'-CH2Cl-DAB, 4'-CH2Cl-DAB, 3'-CH2OH-MAB, 3'-COOH-MAB, 3'-CH3-AB, and 3'-CH2OH-AB, which are still of unknown car

cinogenicity, are suspected to have carcinogenic potency. Twochemicals, 4'-CH2OH-DAB and 3'-CHO-DAB, which were

1655

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GENOTOXICITY OF AZO DYE COMPOUNDS

Table 1 Results ofhepatocyte/DNA repair test on azo dye compounds

ChemicalsAzobenzenesAzB3.3'-(CH3)iAzB4,4'-(CH,)2AzB3,3'-(CHjOH)jAzB4,4'-(CHjOH)zAzB3,3

'-(CH]OAc)]AzB4,4'-(CH2OAc)2AzB3,3'-(CHia),AzB4,4'-(CH2a)jAzBAminoazobenzenesDAB2'-CHj-DAB3'-CHrDAB4'-CHj-DAB2'-CH,OH-DAB3'-CH2OH-DAB4'-CHjOH-DAB2'-CHjOAc-DAB3'-CHjOAc-DAB4'-CHjOAc-DAB3'-CHO-DABDNA

Doserepair(M)10-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-+

io-510-10-+

io-510-10-+

io-510-10-+

io-510-10-+

io-510-10-+

IO-510-10-+

IO'510-10-+

io-510-10-+

io-510-10--t-

io-510-10-+

io-5io—UDS

grains/nucleus0.3

±1.3*-0.3±1.3-0.3±1.4-0.3

±1.5-0.7±1.6-0.6±1.4-0.3±1.4-0.1±1.1-0.3±1.4-0.5±1.2-0.6±1.4-0.4±1.2-0.1±1.4-0.3±1.4-0.2±1.3-0.3

±1.3-0.3±1.3-0.3±1.4-0.3±1.3-0.3±1.3-0.3

±1.4Toxic-0.5

±1.6-0.2±1.1Toxic-0.5

±1.6-0.2±1.1Toxic15.0

±7.65.9±4.3Toxic25.1

±8.67.2±4.3Toxic35.1

±8.322.8±9.9Toxic18.4

±7.23.7±3.9Toxic21.5

±6.515.6±5.4Toxic36.9

±7.812.8±4.9Toxic19.1

±6.111.1±4.4Toxic39.1

±7.910.8±5.2Toxic23.6

±6.819.2±5.3Toxic17.7

±5.44.0±2.930.4±11.014.7±4.96.4±3.7%

of UDSpositivecells10000000000000000000000009450985710094922395941009098901008897959429989757DNAChemicalsrepair4'-CHO-DAB+2'-COOH-DAB+3'-COOH-DAB+3'-CH2CI-DAB+4--CH2C1-DAB+MAB+3'-CHj-MAB+S'-CHjOH-MAB+3'-COOH-MAB+AB+3'-CHj-AB-l_3'-CHîOH-AB+3i2'-(CHj)2-AB+DiaminoazobenzenesDADAB_N'-AcADABN'-AcMADAB+MAMAB_N'-AcMAMAB+N'-AcMA-N-AcMAB+N-2-fluorenylacetamide(positive

control)+Dimethyl

sulfoxide (sol--ventcontrol)Dose(M)io-io-510-10-io-510-10-io-510-io-io-510-10-io-s10-io-io-510-10-io-510-io-io-J10-io-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-io-510-10-io-5io-io—io-510-UDS

grains/nucleusToxic9.9

±3.52.8±2.2Toxic6.4

±2.91.8±1.922.8±7.54.3±3.42.2±1.9Toxic7.9

±5.20.8±1.48.4±4.67.3±5.04.7±3.6Toxic85.7

±9.218.3±7.5Toxic93.7

±11.435.9±6.3Toxic88.4

±8.331.3±7.831.4

±7.720.0±5.96.1±4.0Toxic33.4

±8.51.4±1.9Toxic22.0

±8.92.9±2.553.3±9.612.5

±5.14.0±3.083.5±10.229.5±7.84.8±3.6-0.4

±.5-0.4±.5-0.3±.4-0.1±.4-0.2±.4-0.2±.567.1±12.636.5±6.416.1

±6.9Toxic-0.2

±1.3-0.1±1.369.2±6.338.7±13.820.6±8.3Toxic59.1

±6.017.2±5.987.3

±8.647.3±9.28.3

±3.5-0.6±1.5%

of UDSpositivecells801466897321162268513410094100991009798%481004961510091251009734000000100999400100969510093100100690

* +, induced DNA repair; -, failed to induce DNA repair.* Mean ±SD of triplicate coverslips.

genotoxic, were not carcinogenic in our previous experimentsin which tumorigenic effects in rats were compared for severalaminoazo dyes, including 3'-CH3-DAB or 3'-CH2OH-DAB

(14, 15). However, the negative carcinogenic activities of bothcompounds in rats might be due to the short experimentalperiod (4 months), and in light of the present observations,more extensive studies using longer exposures or differentstrains and species should be studied. Regardless, the findingssupport the concept that the amino group is essential forgenotoxicity and carcinogenicity.

In the DNA repair test, the level of UDS elicited by suchchemicals as 4'-CHO-DAB, 2'-COOH-DAB, 3'-CH2Cl-DAB,or 4'-CH2Cl-DAB was lower than that of other aminoazoben-zenes. 2'-COOH-DAB has been known to have relatively

weaker carcinogenic activities (33). These results, consequently,suggest a consistency between the genotoxicity and carcinogenicity of the aminoazobenzenes. Some parallelism between themutagenic and carcinogenic activities has been observed for azodyes such as 3'-CHj-DAB or 3'-CH3-MAB and their mother

compounds (11).1656

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GENOTOXICITY OF AZO DYE COMPOUNDS

1200-

£ WOO-

§ 600-

¥£

600

400

BTable 2 Genotoxicity, mutagenicity, and carcinogenidty of tested aio dye

compounds

0.1 0.25 0.5 1.0 0.1 0.25 0.5 1.0

«mountof a/o compounds(

Fig. 2. Dose-response curves for mutation of S. typhimurium TA 100 by AzB(A) and 4,4'-(CH2OAc)2AzB (A) in the absence (O) or presence (•)of rat liver

S9.

0.1 0.25 0.5

Amountof cumtn/ocoraraimdstumol/nlme)Fig. 3. Dose-response curves for mutation of S. typhimurium TA98 (A) and

TA 100 (B) by aminoazobenzenes in the absence (O) or presence of rat liver S9.O, 2'-CH2OAc-DAB; •, 4'-CH2OAc-DAB; A, 3'-CH2CI-DAB; A, 3,2'-(CHj)jAB; D, 2'-COOH-DAB; •4'-CHO-DAB.

B

~ 1400-

* 200-

0.1 0.25 0.5 1.0

Anoint of dinlnoozo convounds (pinol/olote)

Fig. 4. Dose-response curves for mutation of S. typhimurium TA98 (A) andTA 100 (A) by diaminoazobenzenes in the absence (O) or presence of rat liver S9.O, DADAB; •,N'-AcMADAB; A, N'-AcADAB; A, MAMAB; D, N'-AcMAMAB; •N'-AcMA-AcMAB.

ChemicalsAzobenzenesAzB3,3'-(CH3)2AzB4,4'-(CH3)2AzB3,3'-(CH2OH)2AzB4,4'-(CH2OH)2AzB3,3'-(CH2OAc)2AzB4,4'-<CH2OAc)2AzB3,3'-(CH2CI)2AzB4,4'-(CH2CI)2AzBAminoazobenzenesDAB2'-CH3-DAB3'-CH3-DAB4'-CH3-DAB2'-CH2OH-DAB3'-CHjOH-DAB4'-CH2OH-DAB2'-CH2OAc-DAB3'-CH2OAc-DAB4'-CH2OAc-DAB3'-CHO-DAB4'-CHO-DAB2'-COOH-DAB3--COOH-DAB3'-CH2Cl-DAB4'-CH2Cl-DABMAß3'-CH3-MAB3'-CH2OH-MAB3'-COOH-MABAB3'-CHj-AB3'-CH2OH-AB3,2'-(CH3)2ABDiaminoazobenzenesD

ADABN'-AcADABN'-AcMADABMAMABN'-AcMAMABN'-AcMA-N-AcMABHPC/DNA

Bacterial mu-repairtagenicity°—

+--———————

—+*————+

+(H)++(17)++(11)++(17)++(17)++(H)++(17)++*+

+ni)++++J1I)++++*+

+Õ11)+++NE"+

+(11)++(11)++(11)++(11)++(11)++(11)++(11)++*-

-*—+*++*__*++'+

—*Carcinogenicity7«77779777+(1,5,6,8)+(5,7)+(6,7)+(1,5,7)+(15)+(12,14)-(15)777^14)7+(3)+(3)—

(14)?7+(5,6,8)+(2)77+(9)79+(4)-(7,28)-(28)+(28)-(28)+(28)+(28)

" Positive data are all on the test with S9 except the chemical marked (*).* Data from this study.' ?, unknown.' NE, not yet examined.

detect one carcinogen and was positive for a non-carcinogen.The basis for the discrepancy between the genotoxicity and themutagenicity on these diaminoazobenzenes as well as the twoazobenzenes is not yet clear. Bacterial mutagenicity may not beas good a predictor of eukaryotic carcinogens, and UDS maynot be related to the mechanistic process of carcinogens, sinceit measures the capacity of the chemical to reach the DNA,whefe it is presumably excised in an error-free fashion.

A correlation between mutagenicity or ability to induce UDSand carcinogenicity have been reported by others for severalazo dye compounds (24, 25, 34). The present findings showthat the hepatocyte DNA repair test, together with the Salmonella/'mutagenicity test, provides a reliable indication of the

potential hazard of azobenzene compounds.

REFERENCES

The results in the DNA repair test with the diaminoazobenzenes were of particular interest. Of the 6 compounds, onlythose which had been reported to be carcinogenic (27, 28) were 2found to be genotoxic in the text. Unlike aminoazobenzenes, .,the presence of an amino group in diaminoazobenzenes wasnot effective for eliciting genotoxicity. An W-acetyl-W -methylgroup may be necessary for the expression of genotoxicity ofdiaminoazobenzenes. In contrast, the mutagenicity test did not

1657

Kinoshita, R. Researches on carcinogenesis of various chemicals. Gann, 30:423-426, 1936.Miller, J. A., and Miller, E. C. The carcinogenic aminoazo dyes. Adv. CancerRes., /: 339-396, 1953.Fujita, S., Adachi, S., and Uesugi, T. Structure-activity study on inductionof hepatic drug metabolizing enzymes by azo compounds. Biochem. Phar-macol., 30:1147-1150, 1981.Sasaki, T., and Yoshida, T. Experimentelle erzeugung des Leber-carcinomasdurch Futterung mit O-Aminoazotoluol. Virchows Arch. Pathol. Anal. Phys-iol. Klin. Med. 29: 175-200, 1935.

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GENOTOXICITY OF AZO DYE COMPOUNDS

5. Miller, J. A., and Bauman, C. A. The carcinogenkity of certain azo dyesrelated to /wlimethylaminoazobenzene. Cancer Res., 5:227-233,1945.

6. Miller, J. A., and Miller, E. C. The carcinogenicity of certain derivatives of/Miimethylaminoazobenzene in the rat. J. Exp. Mod.. 87:139-156,1948.

7. Miller, J. A., Miller, E. C., and Finger, G. C. Further studies on thecarcinogenicity of dyes related to 4-dimethylaminoazobenzene. The requirement for an unsubstituted 2-position. Cancer Res., 17: 387-398, 1957.

8. Miller, J. A. Carcinogenesis by chemicals: an overview—G. H. A. ClowesMemorial Lecture. Cancer Res., 30: 559-576, 1970.

9. Odashima, S., and Hashimoto, Y. Carcinogenicity and target organs ofmethoxyl derivatives of 4-aminoazobenzene in rats. II. Effect of variousconcentration of 3-methoxy- and 2,5-dimethoxy-4-aminoazobenzene in thediet. Gann, 61:153-160,1970.

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1986;46:1654-1658. Cancer Res   Hideki Mori, Yukio Mori, Shigeyuki Sugie, et al.  

/Mutagenicity TestSalmonellaCompounds in the Hepatocyte/DNA Repair Test and the Genotoxicity of a Variety of Azobenzene and Aminoazobenzene

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