115

Mutatwn Research, 260 (1991) 115-119 © 1991 Elsevier Science Pubhshers B.V. 0165-1218/91/$03 50 ADONIS 0165121891000863

MUTGEN 01648

DNA lesion in rat hepatocytes induced by in vitro and in vivo exposure to glyoxal Hitoshi Ueno

1 Katsuhiko

Nakamuro

1, Y a s u y o s h i

Sayato a and Shoji Okada 2

1 Dwtston of Envtronmental Health, Faculty of Pharmaceutwal Sctences, Setsunan Umverstty, 45-1 Nagaotoge-cho, Hlrakata, Osaka 573-01 (Japan) and 2 Department of Radtobtochemtstry, School of Pharmaceutwal Scwnces, Umverstty of Shtzuoka, 395 Yada, Shtzuoka 422 (Japan) (Recewed 20 July 1990) (Revision received 23 October 1990) (Accepted 29 October 1990)

Keywords Glyoxal; Alkaline elutlon; DNA single-strand breaks, Rat hver; Hepatocytes, Genotoxaclty, Ozonat,on products

Summary The alkaline elution technique was applied to measure the damage of rat hepatic DNA following exposure to glyoxal. DNA single-strand breaks were induced after exposure of primary-cultured hepatocytes to 0.1-0.6 m g / m l glyoxal for 60 min, while no DNA cross-link was observed. Single-strand breaks were also detected in livers of rats within 2 h following a single oral exposure at 200-1000 m g / k g body weight, and the frequency of the breaks reached a maximum around 9 h after exposure. The breaks were almost fully repaired 24 h after exposure to any dose. However, hardly any DNA lesions were detected in other tissues following exposure to 1000 m g / k g glyoxal. Thus, the present results indicate that glyoxal causes DNA single-strand breaks in rat hepatocytes following in vitro and in vivo exposure.

Glyoxal is an a-keto aldehyde present widely in the environment: in ozonated drinking water (Glaze et al., 1989; Ueno et al., 1989), in photooxidants of atmospheric aromatxc hydrocarbons (Tuazon et al., 1984), m cigarette smoke (MoreeTesta and Saint-Jalm, 1981), and in foods such as heated glucose (Kasai and Nishimura, 1986) and

Correspondence: Dr Y. Sayato, Division of Envaronmental Health, Faculty of Pharmaceutical Soences, Setsunan University, 45-1 Nagaotoge-cho, Hlrakata, Osaka 573-01 (Japan) Abbrevtatwns. MMS, methyl methanesulfonate; HEPES, N-2hydroxyethylp~perazlne-N'-2-ethanesulfonic aod, PBS, phosphate-buffered sahne.

coffee (Kasat et al., 1982). This aldehyde is directly mutagenic in Salmonella as described previously (Sayato et al., 1987). Its administration to rats induced unscheduled DNA synthesis and ornithlne decarboxylase activity in stomach mucosa, showing potential tumor-initiating and -promoting activity (Furihata et al., 1985). Although glyoxal is thus a possible carcinogen broadly distributed in the environment, there have yet been few studies of its genotoxicologtcal effect on mammals. In the present work, we applied the alkaline elution technique (Kohn et al., 1981) to examme the formation and repair of lesions of rat hepatic DNA following in vitro and in vivo exposure to glyoxal, and to detect both DNA single-strand

116 breaks and cross-hnks.

DNA-interstrand

or DNA-protein

Materials and methods Chemtcals Glyoxal (ethanedial, as trimeric dihydrate, CAS No. 107-22-2) was obtained from Sigma Chemical Co.; methyl methanesulfonate (MMS, CAS No. 66-27-3) from Tokyo Kasel Kogyo; proteinase K from Merck; tetrapropyl a m m o n i u m hydroxide from Eastman K o d a k Co.; and 3,5-dlaminobenzoic acid from Aldrich Chemical Co. Glyoxal solunon was freshly prepared before use by dissolving the trimeric dihydrate in distilled water at 4 0 - 5 0 ° C to avoid polymerization. In vitro exposure of primary-cultured hepatocytes Hepatocytes were isolated from male Sprague-Dawley rats (140-150 g) by the collagenase perfusion technique of Bery and Friend (1969) as described by Bradley and Sina (1984). The hepatocytes were dispensed into 90-mm bacteriological petri dishes at 105 c e l l s / m l in 11 ml of Wllllam's medium E (Flow Laboratories) containing 10 m M N-2-hydroxyethylpiperazlneN'-2-ethanesulfonlc acid (HEPES), 100 I U / m l penicllhn, 100 /~g/ml streptomycin, 0.25 /~g/ml amphotericin B and 10% fetal calf serum. Glyoxal was added in volumes of 0.02-0.1 ml and mixed, and the dishes were incubated at 37°C in 5% CO 2 for 60 min. After incubation, the cells (1.0 ml) were tested for their viablhty by trypan blue exclusion, and the remains were loaded onto 2 /~m pore size, 25 m m diameter polycarbonate filters (Nucleopore Corp.) for alkaline elutlon assays, washing out the dishes with 10 ml of ice-cold phosphate-buffered saline (PBS). In vwo exposure of rats Five-week-old male S p r a g u e - D a w l e y rats (Japan SLC Co.), weighing approximately 150 g, were starved overnight and then admimstered 1.0 ml of a glyoxal solution by gastric intubation. After 1-24 h of exposure, the animals were killed, the livers perfused with saline with a 50-ml syringe through the portal vein, and the tissues rapidly removed and placed in ice-cold 0.024 M E D T A 0.075 M NaC1 solution (pH 7.5) ( E D T A - N a C I ) to

avoid degradation of nuclear D N A by DNase following cell crush. The liver was briefly minced and then homogenized using a loosely fitting Potter-Elvehjem homogenizer. After centrlfugatlon at 50 × g for 1 min to remove large fragments, the supernatant was removed, centrifuged at 700 × g for 1 min, and then discarded. The pellet was then resuspended in E D T A - N a C 1 and centrifuged again at 700 × g for 1 man. This washing of the nuclear pellet was repeated at least 4 times. The liver nuclei were diluted in E D T A NaC1 and applied to the filters for alkaline elutlon assays. Finely minced kidney, spleen, pancreas and lung were homogenized with a hand Teflonglass homogenizer and centrifuged at 100 × g for 2 - 3 mln. An aliquot of supernatant containing crude nuclei was then applied to the filter. Bone marrow cells were prepared according to the method of Parodi et al. (1982). Approximately 1 × 106 of these nuclei and cells were loaded onto the filters. Alkahne elutton assays Alkaline elunon assays were performed by the procedure described by Slna et al. (1983) with modifications. The cells or nuclei were lysed on the filter with 1.5 ml of lysis solution (pH 9.6) containing 1 m g / m l proteinase K for 60 mln in the dark, and D N A was eluted with 30 ml of tetrapropyl a m m o n i u m hydroxide elutlng solution (pH 12.1) at a flow rate of 0.035 m l / m i n in the dark. The amounts of D N A both eluted and remaining on the filter were determined with 3,5-diamlnobenzoic acid according to the method of Bradley et al. (1982). The elutlon rate constant K (ml-1) was calculated as described by Parodi et al. (1982). To detect both D N A single-strand breaks and D N A cross-links in primary-cultured hepatocytes, the glyoxal-treated cells were assayed either directly or after exposure for 10 min, immediately before elution, to I m M MMS (an reducer of single-strand breaks) at 37°C as described by Brambilla et al. (1985). The cultured hepatocytes treated with MMS were assayed without digestion of the lysate by proteinase K to detect possible DNA-protein cross-links. The apparent frequencies of single-strand breaks and cross-links were calculated from the equations of Ewig and Kohn

117 TABLE 1

i0 0

APPARENT FREQUENCY OF DNA LESIONS BY GLYOXAL IN RAT HEPATOCYTES a

ii

Glyoxal (mg/ml)

Apparent lesion frequency ( × 10- 9 Da) Single-strand breaks

Cross-links

0.1 03 06

4 13_+0 23 122 _+07 23.4 -+14

- 0 44_+0 12 -281+078 - 5 2 4 + 1 38

Mean values and SD (n = 3) of glyoxal-induced lesion frequency were calculated from the data of the fraction of DNA remmmng on the filter 12 h after the elution shown in Fig 1, according to the equations of Ewlg and Kohn (1978)

i0-2 0

3

6

9

12

0

3

6

9

12

flours of Elution

Fig 1 Profdes of alkaline elution performed without (A) and with (B) exposure of rat hepatocytes to methyl methanesulfonate following treatment with glyoxal. Following mcubaUon of hepatocytes without (©) or wnh 0 1 (@), 0 3 (A) and 0 6 (m) mg/ml glyoxal for 60 nun, alkahne eluuon assays were performed with (A) or without (B) dlgesUonof the lysate with protemase K Elutmn profiles (B) of cells exposed to 1 mM MMS are represented by dotted lines The elutlon curves represent the average of trlphcate eluuon runs for each sample

(1978), the frequency of single-strand breaks produced by 1 m M M M S being 3 . 4 × 1 0 -9 D a (Brambtlla et al., 1985).

Results D N A damage tn primary-cultured hepatocytes F o r the m e a s u r e m e n t of c o n c u r r e n t D N A single-strand breaks a n d D N A - i n t e r s t r a n d or D N A - p r o t e i n cross-links, alkaline ehition assays were performed b o t h with a n d without exposure of the hepatocytes to M M S following t r e a t m e n t with glyoxal. As shown in Fig. 1A, 60-mln exposure of hepatocytes to 0.1, 0.3 a n d 0.6 m g / m l glyoxal resulted m a n increased rate of alkahne elution of D N A from the filters with increasing dose, i n d i c a t i n g the i n d u c t i o n of single-strand breaks. F r o m the elutlon patterns in the MMS-exposed cells, neither reduction of the elution rate n o r a d o s e - d e p e n d e n t increase of the rate resulted from the glyoxal t r e a t m e n t (Fig. 1B). As shown in

T a b l e 1, however, the calculated a p p a r e n t lesion frequency for cross-links was a negative n u m b e r with a n y dose of glyoxal, m e a n i n g that less D N A was retained o n the filter in glyoxal-treated t h a n m control cells, a n d accordingly there was n o i n d u c t i o n of cross-links. The frequency of singlestrand breaks was d o s e - d e p e n d e n t (Table 1). The m e a n vlabxhty of hepatocytes which was 93 + 3% after isolation r e m a i n e d 88 + 2% even following exposure to a m a x i m u m dose of 0.6 m g / m l glyoxal for 60 min.

D N A smgle-strand breaks m rat hver following m vtvo exposure As glyoxal d o m i n a n t l y i n d u c e d D N A singlestrand breaks in hepatocytes in vitro, the breaks m rat hver following m vivo exposure to the comp o u n d were measured. D N A lesions were detected in liver within 2 h of a single oral dose of 200, 500 or 1000 m g / k g b o d y weight a n d reached a maxim u m a r o u n d 9 h after exposure, r e t u r n i n g nearly to control levels 24 h after exposure to a n y dose (Fig. 2A). Fig. 2B d e m o n s t r a t e s the dose-related increase in the elution rate c o n s t a n t of D N A from the hver 9 h after exposure to glyoxal. The alkaline elution of D N A from other tissues following exposure of rats to 1000 m g / k g glyoxal resulted in httle r e d u c t i o n of single-strand breaks, though a modest increase in the e l u t i o n rate constant was measured m spleen (Table 2).

Discussion The results of the present study d e m o n s t r a t e that glyoxal causes D N A lesions b y single-strand

118 TABLE 2

slon. It has been reported that this substance causes cyclic adduct formation of guanine base in DNA (Demoulin et al., 1978; Kasai et al., 1984). Glyoxal has a high reactivity and also yields adducts with amino acids possessing amino, imino or sulfhydryl residues in in vitro systems (in press). It is, however, noteworthy that DNA lesions in primary-cultured hepatocytes were observed even when exposed to glyoxal in an amino acid-rich medium containing fetal calf serum. The present results suggested that glyoxal does not induce total DNA-interstrand and DNA-protein cross-links in hepatocytes. Methylglyoxal, a glyoxal derivatwe where a hydrogen is replaced by a methyl group, produced both slight induction of single-strand breaks and cross-links m the cells (unpublished data). This derivative was also shown to induce cross-links but few single-strand breaks in Chinese hamster ovary cells (Brambilla et al., 1985). In the case of glyoxal, the induction of single-strand breaks in the pyloric mucosa of rat stomach after 50-550 m g / k g p.o. exposure has been reported (Furihata et al., 1989). Glyoxal administration also gave an increase in the incidence of adenocarcinomas in the pylorus of the glandular stomach of rats pretreated with N-methyl-N'-

E L U T I O N R A T E C O N S T A N T S K O F D N A F R O M TISSUES OF RATS 9 h A F T E R E X P O S U R E T O 1000 m g / k g GLYOXAL T~ssue

Liver Kadney Spleen Pancreas Bone marrow Lung

N u m b e r of experiments

4 3 2 2 2 2

Elutmn rate constant a B / A K ( m l - 1) X 10 3 Control group (A)

Glyoxaltreated group (B)

2 2 2 6 2 4

22.4 * 2.68 4.58 7.00 2.50 3.89

72 99 89 18 32 21

82 09 1.6 11 11 09

Mean values are g~ven. * S,gmflcantly different from control group by Student's t-test, p < 0.01.

a

breaks in hepatocytes following in vitro exposure and also in livers of rats after oral exposure. Since the reduction in the viability of cultured hepatocytes exposed to glyoxal was negligible, the lysosomal DNA hydrolysis resulting from cell lysis was unlikely to be responsible for this DNA le-

O~l

i

l

l

i

I

I

I

I

I

I

I

I

0

3

6

9

12

15

18

21

24

0

200

500

1000

llours After Treatment

Dose (mg/kg)

F,g. 2 Dependence of the eluUon rate constant K of D N A from rat hver on tLrne interval after exposure to glyoxal (A) and on its dosage 9 h after exposure (B). Dose of glyoxal ( m g / k g body weight): 200 (e), 500 (A), 1000 (11); control (©) Each point represents the m e a n value of at least 3 independent experiments, bars, SD

119 nitro-N-nitrosoguanidine and sodium chloride ( T a k a h a s h i e t al., 1989). On the basis of our results, glyoxal must be g e n o t o x i c i n r a t liver, h o w e v e r , n o a n i m a l s t u d y o n its c a r c i n o g e n l c i t y h a s y e t b e e n r e p o r t e d . A long-term test would be valuable to evaluate the r i s k o f g l y o x a l t o m a n b e c a u s e o f its w i d e p r e s e n c e in the environment. Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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Kasax, H , and S. Nlstumura (1986) Hydroxylatlon of guamne m nucleosldes and DNA at the C-8 posmon by heated glucose and oxygen ra&cal-formmg agents, Environ. Health Perspect., 67, 111-116. Kasal, H , K. Kumeno, Z Yamalzurm, S. Nlshimura, M Nagao, Y. Fuj~ta, T Suglmura, H. Nukaya and T. Kosuge (1982) Mutagemclty of methylglyoxal m coffee, Jpn J Cancer Res. (Gann), 73, 681-683. Kasal, H., H. Hayarm, Z. Yamalzurm, H. Saxt~ and S Nlstumura (1984) Detection and ldentlficaUon of mutagens and carcmogens as their adducts with guanosme derivatives, Nucleic Aods Res., 12, 2127-2136. Kohn, K.W., R.A G. Ewlg, L.C. Erickson and L A Zwelhng (1981) Measurement of strand breaks and cross-hnks by alkahne elutlon, m E C. Frledberg and P.C. Hanawalt (Eds.), DNA Repmr: A Laboratory Manual of Research Procedures, Vol 1, Marcel Dekker, New York, pp. 379-401. Moree-Testa, P, and Y. Saint-Jalm (1981) Deterrmnatlon of a-dlcarbonyl compounds m cigarette smoke, J Chromatogr., 217, 197-208 Parodi, S., M Pala, P Russo, A. Zunino, C. Balbl, A Alblm, F Valeno, M R Clmberle and L Santl (1982) DNA damage m hver, kadney, bone marrow, and spleen of rats and rmce treated with commercial and purified amhne as determined by alkahne elutlon assay and sister chromatld exchange induction, Cancer Res., 42, 2277-2283 Sayato, Y., K. Nakamuro and H. Ueno (1987) Mutagemoty of products formed by ozonatlon of naphthoresorcinol m aqueous solutions, Mutation Res, 189, 217-222. Sma, J F., C.L. Bean, G.R Dysart, V.I Taylor and M.O Bradley (1983) Evaluation of the alkaline elutlon/rat hepatocyte assay as a pre&ctor of carclnogemc/mutagemc potentml, Mutation Res., 113, 357-391. Takahasla, M , H Okarmya, F. Furukawa, K Toyoda, H Sato, K Imalda and Y. Hayasha (1989) Effects of glyoxal and methylglyoxal admamstratlon on gastric carcmogenesss m Wlstar rats after mltlaUon with N-methyl-N'-mtro-Nrmrosoguamdlne, Carcinogenesis, 10, 1925-1927. Tuazon, E.C., R. Atlonson, H.M. Leod, H W. Blermann, A M. Winer, W.P L Carter and J.N. Pltts Jr. (1984) Yields of glyoxal and methylglyoxal from the NOx-alr photooxadatlons of toluene and m- and p-xylene, Enwron. SCL Technol, 18, 981-984 Ueno, H., T Segawa, K Nakamuro, Y. Sayato and S. Okada (1989) Mutagemoty and ldenuflcatlon of products formed by aqueous ozonation of hurmc acids of different ongms, Chemosphere, 19, 1843-1852.