Genotoxicity testing of chloramphenicol in rodent and human cells

65

Mutatton Research, 260 (1991) 65-72 © 1991 Elsevier Science Pubhshers B V 0165-1218/91/$03 50 ADONIS 016512189100080L

MUTGEN 01642

Genotoxicity testing of chloramphenicol in rodent and human cells Antonietta Martelli 1, Francesca Mattioli 1, Giorgio Pastorino 2, Luigi Robbiano 1, Alessandra Allavena 1 and Giovanni Brambilla 1 1 Instttute of Pharmacology, Umverstty of Genoa and 2 Ftrst Surgtcal Dtvzswn of St Martmo Hospital, I-16132 Genoa (Italy)

(Received 26 June 1990) (Revision recewed 12 September 1990) (Accepted 21 September 1990) Keywords Chloramphemcolgenotoxaoty assessment; Rat hepatocytes, Human hepatocytes, V79 cells; DNA damage; DNA repaxr,

Mutagemc acttvity, Micronucleus test

Summary The results of this work, carried out to extend the limited information at present available on the genotoxic potential of chloramphemcol (CAP), indicate that in millimolar concentrations this antibacterial agent produced a minimal amount of D N A fragmentation in both V79 cells and metabolically competent rat hepatocytes. Moreover, a level of DNA-repair synthesis indicative of a weak but positive response was detected in primary cultures of liver cells obtained from 2 of 3 human donors, and a borderline degree of repair was present in those prepared from rats. The promutagenic character of CAP-induced D N A lesions was confirmed by a low but significant increase in the frequency of 6-thioguanine-resistant clones of V79 cells, which, however, was absent when the exposure was done in the presence of co-cultured rat hepatocytes. Finally, oral administration to rats of ½ LDs0 CAP did not increase the incidence of either micronucleated polychromatic erythrocytes or micronucleated hepatocytes. Taken as a whole these findings suggest that CAP should be considered a compound intrinsically capable of producing a very weak genotoxic effect, but only at concentrations about 25 times higher than those occurring in patients treated with maximal therapeutic dosages.

Chloramphenicol (CAP) is a broad-spectrum antibiotic employed to treat bacterial, rickettsial and chlamydial diseases. Recently the use of CAP has increased because of the emergence of ampicillin-reslstant strains of H a e m o p h d u s m f l u e n z a e and because of its effectiveness in anaerobic infections of the central nervous system. CAP belongs to the family of nitroaromatic compounds that are asso-

Correspondence' Dr. Glovanm Brambdla, Istltuto dl Farmacologm dell'Umvers~th, Vlale Benedetto XV 2, 1-16132 Genoa (Italy)

ciated with mutagemcity and carcinogemcity (Rickert, 1985; IARC, 1987a; Rosenkranz, 1987). As pointed out by Rosenkranz (1988) in a recent review, because it was developed before the present scientific and regulatory climate, CAP has not been systematically tested for genotoxicity and, as summarized by I A R C (1987b), the few available data are contradictory. CAP was found to reduce chromosomal aberrations in plants, in cultured pig and h u m a n lymphocytes, and in bone-marrow cells of mice treated in vivo. In contrast, CAP was not D N A - d a m a g i n g and mutagenic in bacteria, and it did not induce sister-chromatid exchanges in cul-

66 tured human lymphocytes, chromosomal aberrations in bone-marrow cells of rats treated in vivo, and mutations in Drosophila. Results provided by the dominant lethal mutation assay in mace were discordant. It must be pointed out that CAP has not been tested for its abihty to induce DNA-repair synthesis and gene mutations in cultured mammalian cells. CAP was classified by the International Agency for Research on Cancer (IARC, 1987a) among chemicals possibly carcinogenic to humans (limited evidence of carcinogemcity in humans and inadequate evidence of carcinogenicity in animals). Recently IARC (1989) has revised the CAP carcinogenicity data and, considering the unusual incidence of leukaemia following CAP-induced aplastic anaemia and bone-marrow depression, concluded that CAP is probably carcinogenic to humans. In this paper CAP genotoxlclty was evaluated in vwo with the rnlcronucleus assay in liver and bone marrow of rats, and in vitro by measuring DNA fragmentation and DNA-repair synthesis in V79 cells and rat hepatocytes. Because of the possibility that extrapolation to humans of results obtained in other species can be distorted by differences in the metabolic activation/detoxificat~on of this antibacterial agent, we deemed it of interest to extend the assessment of CAP genotoxicity to human hepatocytes. Materials and methods Chemtcals

Chloramphemcol (CAP) was purchased from Serva Feinbiochemica (Heidelberg, F.R.G.); collagenase type IV, DL-buthionine-S,R-sulfoximine (BSO), N-nitroso-N-methylurea (NMU), and 6thioguanine (TG) from Sigma Chemical Co. (St. Louis, MO, U.S.A.); Williams' medium E (WME), and a-modified minimal essential medium minus purines and pynmidines (a-MEM) from Flow Laboratories (Milan, Italy); fetal bovine serum from Boehringer (Mannheim, F.R.G.); N-nitrosodimethylarmne (DMN), Schiff reagent, and May-Griinwald-Giemsa stare from E. Merck ( D a r m s t a d t , F.R.G.); [methyl-3H]thymidine (specific activity 25 Ci/mmole) from Amersham International (Amersham, U.K.). All other chemicals were reagent grade.

In vitro assays Cell culture and treatment. V79 cells (NIH, Coriell Institute for Medical Research, Camden, N J, U.S.A.), an established line from Chinese hamster lung, were cultured in a-MEM supplemented with 5% fetal bovine serum. Ethanol was used as a solvent for CAP and was added at the same concentration m control cultures. For the determination of cytotoxicity cells were seeded in 25-cm2 plastic flasks and after 24 h were exposed to CAP for 1 or 20 h in serum-free medium. Cell viability was measured immediately after exposure either by trypan blue dye exclusion or by cloning efficiency. In the trypan blue assay, at least 1000 cells/sample were examined. In the colony-formation assay, cells were trypsinised at the end of treatment and inoculated in plastic d~shes (200 cells/dish); after incubation for 7 days, colomes were fixed, stained, and counted. For the assay of DNA-damaging activity, 24 h after seeding V79 cells were labelled with 0.1 /~Ci/ml of [methyl-3H]thymidine for 24 h. Cells were then exposed to CAP for 1 h in non-radioactive serum-free medium and assayed for D N A fragmentation immediately afterwards. Rat hepatocytes were isolated from SpragueDawley male albino rats (200-250 g) by collagenase perfusion, as described by Williams et al. (1982), and used when cell viability, as assessed by trypan blue exclusion, was > 80%. H u m a n hepatocyte suspensions were prepared from fragments of human liver discarded during the course of prescribed surgery essentially according to Strom et al. (1982). Details of donors of liver samples are reported in Table 1. Isolated rat and human hepatocytes were suspended in WME supplemented with 10% fetal bovine serum and gentanucin (50/~g/ml), and aliquots of these suspensions were plated in plastic dishes as follows: 2 × 10 6 cells in 60-mm uncoated dishes for D N A fragmentation assays, and 1 × 10 6 cells in 35-mm dishes coated with rat tail collagen for determination of cell viability and DNA-repair synthesis. After attachment for 3 h cells were incubated for 20 h with CAP m serum-free medium. D M N was used as the positive control to verify the capability of hepatocytes to activate procarcinogens. To produce GSH depletion hepatocytes were treated with

67 TABLE 1 DETAILS OF PATIENTS FROM WHOM LIVER SAMPLES WERE OBTAINED FOR THE ISOLATION OF H EPATOCYTES Donor Iniuals Sex Age (years) Indication for surgery Blhrubm (mg/ml) Alkahne phosphatase (U/l) AST a (U/l) ALT (U/l) T-GT (U/l) CPK (U/l) LDH (U/l) Cell vlabthty (%)

1

2

3

CC F 47 Massive antrahepatic lltlUasxs

AB M 46 Cavernous haemangtoma

A.T. F 65 Hepatxc metastasis of colon carcinoma

9.4

10

06

152 225 304 30 -

27 27 32 -

721 21 32 246 32 349

75

65

89

a AST, aspartate armnotransferase, ALT, alanme armnotransferase, T-GT, gamma-glutamyltransferase, CPK, creatlne phosphokmase; LDH, lactate dehydrogenase.

1 m M BSO; this treatment was simultaneous with exposure to CAP. In these conditions cellular G S H was reduced to approximately 30% of the control value without any significant effect on cell viability. D N A fragmentation and repair were assayed immediately after exposure to the test c o m p o u n d . Determmatwn of DNA fragmentatton and repair. The presence of D N A fragmentation (single-strand breaks a n d / o r alkali-labile sites) was detected b y the alkaline elution technique ( K o h n et al., 1976) with the minor modifications previously described (Brambilla et al., 1989). Single-strand D N A eluted f r o m the filter at a p u m p speed of 0.13 m l / m i n was collected in 1.3-ml fractions at 10-min intervals. The D N A content of each fraction and that remaining on the filter was determined by a microfluorimetric procedure first developed by Kissane and Robins (1958) and modified b y Brambilla et al. (1978). The induction of D N A - r e p a i r

synthesis was evaluated according to Wllhams et al. (1982). Determmatton of mutagemc acttvlty. T h e quantitative assay for induced frequency of 6thioguanlne-resistance ( T G r) cells was a modification (Cajelli et al., 1987) of that described by M y h r and Di Paolo (1978). The duration of exposure to the test chemical was 1 h. At the end of the 6-day period of m u t a n t expression, the fraction of T G r cells in the population was determined from 10 dishes (100 mm) by counting m u t a n t colomes 6 - 7 days after the seeding of 3.5 x 105 cells per dish. F o r selection of mutants, 6-thioguanine was added 2 h after seeding to give a final concentration of 1 0 / ~ g / m l . Mutagenicity in the presence of a metabofic system represented by rat hepatocytes was determined according to the protocol of L a n g e n b a c h et al. (1978). A p p r o x i m a t e l y 1 x 106 viable hepatocytes were added to a 24-h culture of V79 cells, growing in W M E supplemented with 10% fetal bovine serum in a 75-cm 2 flask, so that the ratio of hepatocytes to V79 cells was about 1 : 1. The duration of exposure to the test chemical was 20 h. In vtvo micronucleus test on liver and bone marrow cells Fifteen S p r a g u e - D a w l e y male albino rats (200-250 g) were used. T h e y were r a n d o m l y divided into 3 groups of 5 rats each, and treated as follows: group A, controls; group B, 1250 m g / k g of C A P administered by gavage in a single dose; group C, 10 m g / k g of D M N in a single i.p. dose. The procedure used to evaluate micronucleated hepatocytes was that described by Tates et al. (1980). A 2 partial h e p a t e c t o m y was performed 20 h before treatment; fiver and b o n e - m a r r o w cells were isolated from the same rat 48 h after the administration of the test c o m p o u n d . The liver was perfused as described before; hepatocytes were washed 3 times and resuspended in H a n k s ' solution at a concentration of 1 × 106 cells/ml. T w o drops of the cell suspension were put on a microscope sfide, fixed, and stained as described b y Tates et al. (1980) with the following m i n o r modifications. Hepatocytes were fixed for 90 rain in methanol : acetic acid : 35% formalde-

68 TABLE 2 RELATIVE SURVIVAL POSURE TO CAP

OF

V79

CELLS

AFTER

CAP

R e l a t w e s u r v i v a l as m e a s u r e d b y

(mM)

Plating efhctency

Trypan blue exclusion

1-h exposure a

20-h exposure b

1-h exposure b

20-h exposure

(0.89 5:0 05) 0.97 + 0.03 0.86 5:0.07 0.805:0.01 0 19 0

(0 93) 0 99 0 90 0.54 0 -

(0.90) 1 02 102 1.00 0.67

(0.95) 0.97 097 0 88 0

0 0 5 10 20 4.0 8.0

femur of the same rat in 3 ml of fetal bovine serum, according to the method described by Schmid (1975). Slides were prepared by centrifuging ahquots of a 1 : 1 0 diluted cell suspension at 500 rpm for 10 min in a 'Cytospin 2' (Shandon) centrifuge; they were mr-dried and stained according to Cole et al. (1979). The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal; the ratio of normochromatic to polychromatic erythrocytes was also evaluated.

EX-

Results a D a t a a r e the m e a n s + S D o f 3 i n d e p e n d e n t assays. V a l u e s m p a r e n t h e s e s i n d i c a t e the f r a c t i o n o f v i a b l e cells m c o n t r o l s . R e l a t w e s u r w v a l w a s c a l c u l a t e d f r o m t h e r a U o ( f r a c t t o n of w a b l e cells m t r e a t e d c u l t u r e s ) / ( f r a c t l o n o f viable cells m control cultures) b D a t a are the m e a n s o f 2 i n d e p e n d e n t a s s a y s .

Cytotoxtctty The data listed in Table 2 show that 1-h exposure of V79 cells to CAP in serum-free medium produced a dose-dependent reduction of plating efficiency at concentrations ranging from 0.5 to 8 mM. The cytotoxic effect was increased by prolonging the treatment to 20 h. For both exposure times, the reduction in the fraction of surviving cells was lower when measured by trypan blue exclusion, which shows alterations of the cell membrane related to immediate cell death instead of damage to the reproductive capacity. Primary cultures of both rat and human hepatocytes displayed a greater sensitivity to CAP toxicity, the total disappearance of viable trypan

hyde ( 8 5 : 5 : 1 0 ) , rinsed in distilled water for 5 rain, incubated in 1 N HCI at 60 ° C for 7 rain, and then stained at room temperature in Schiff's reagent for 90 min. The slides were washed with 3 × 2-rain changes of sulphite solution, and for 5 min in tap water; finally, they were dehydrated, rinsed in xylol and mounted in Eukitt. Micronuclei were scored in intact hepatocytes according to Tates et al.'s (1980) suggestions. Bone-marrow cells were isolated from a single

TABLE 3 RELATIVE SURVIVAL OF RAT AND HUMAN Treatment

HEPATOCYTES

BSO -

Human hepatocytes b BSO +

Donor I

Donor 2

BSO-

a Data GSH ratm b Data

TO CAP

R e l a t i v e s u r w v a l of Rat hepatocytes a

Control C A P 0.5 m M CAP 1 0 mM C A P 2.0 m M C A P 4.0 m M D M N 5.0 m M

AFTER 20-h EXPOSURE

(0 97 4- 0 03) 0.974-0.02 0.95 4- 0 07 0 . 8 0 4 - 0 09 0 0.99 4- 0.02

(0 98 4- 0 05) . . 0 94 + 0 04 0.71 4-0 06 0 0.97 + 0.03

BSO+

(0.94) . 1.03 0 83 1.02

BSO-

(0.90)

(0 91) .

. 1 03 0 86 -

. 0.97 0 79 0 0 98

Donor 3 BSO+

BSO-

BSO +

(0.88)

(0.98)

(0.98)

. 0.97 0.75 0 -

0.99 0.99 0 0.97

0 99 0.93 0 -

a r e the m e a n s + S D o f 3 i n d e p e n d e n t a s s a y s c a m e d o u t m t h e a b s e n c e o r m the p r e s e n c e o f 1 m M B S O e m p l o y e d to r e d u c e d e p l e t i o n V a l u e s m p a r e n t h e s e s m d t c a t e the f r a c u o n o f v m b l e cells m c o n t r o l s . R e l a t i v e s u r v i v a l w a s c a l c u l a t e d f r o m the ( f r a c t i o n o f viable cells m t r e a t e d c u l t u r e s ) / ( f r a c t a o n o f vaable cells m c o n t r o l c u l t u r e s ) a r e the m e a n s o f 2 c u l t u r e s p e r d o n o r .

69 TABLE 4 D N A F R A G M E N T A T I O N IN V79 CELLS A N D RAT H E P A T O C Y T E S Treatment

N u m b e r of samples

% D N A eluted from the filter ( m e a n + SD)

Elutton rate over controls (Kt_Kc) a

Slgmficance level ( p ) b

V79 cells (1-h exposure) Control CAP 1.0 mM CAP 2.0 mM CAP 4.0 mM

8 6 10 4

5 6 56.2+ 7 957.5 5-

0.8 0.7 3.3 0.9

( K c = 0 0044) 0 0005 0 0019 0 0016

> 0 10 > 0 10 < 0 05

18.9+ 0 9 20 8 5- 1.6 2005- 1.7 22 0 5- 3.5 63 5 5-15.1

( K c = 0.0161) 0.0018 00011 0 0030 0 0614

< > < <

19.5 5- 0 6 18.85- 1 2 27.3 5- 4 8 67.3 5-16 6

( K c = 0 0167) 0.0007 0.0078 0.0693

> 0 10 < 0.05 < 0 001

Rat hepatocytes (20-h exposure) Control CAP 0.5 mM CAP 1.0 mM CAP 2.0 mM D M N 5.0 mM

11 6 8 10 8

0 05 0.10 0.02 0.001

Rat hepatocytes wtth 1 m M BSO (20-h exposure) Control CAP 1.0 mM CAP 2.0 mM D M N 5.0 mM

4 3 7 6

a Kt Is the elutlon rate of D N A from treated cultures, and K c ts the elutmn rate of D N A from control cultures. b The staUsUcal s~gmficance ( p ) of the difference from controls m the percentage of eluted D N A was evaluated according to the Wdcoxon 2-sample (2-taded) test (Rtimke and De Jonge, 1964).

blue-excluding cells being induced by a 20-h treatment with a concentration (4 mM) 2 times lower than that required to produce the same effect in

V79 cells (Table 3). BSO-induced G S H depletion did not influence the hepatocyte response to a significant extent.

TABLE 5 D N A - R E P A I R SYNTHESIS IN RAT A N D H U M A N H E P A T O C Y T E S P R I M A R Y C U L T U R E S A F T E R 20-h E X P O S U R E TO CAP Treatment

N u m b e r of net nuclear grains (mean 5- SD) a Rat

Control CAP05mM CAP1.0mM CAP 2.0 mM DMN50mM BSO1.0mM CAP 1 mM +BSOlmM CAP 2 mM +BSOlmM

- 0 . 1 + 51 - 1 . 6 + 4.1 +0.7+ 58 + 2 . 4 + 5.0 +20.6___12.5 - 0 . 4 + 5.7 -0.15- 40 +2.3+

(10) (4) (24) (29) ** (85) ** (13) (10)

5.7 ( 3 0 ) * *

Donor I

Donor 2

D onor 3

- 0 . 3 + 2.0 (2) +4.4+ 7.4(50)** + 1 0 . 3 + 7.1 (76) ** +32.3-t-22.8(92) ** -01_ 0.8 (4)

1.5+ 4.3(22) 1.6+ 4.5(18) 2.1+ 3.9(29) 6.7+10.9 (42) ** 14.5_+ 8 7 ( 8 6 ) ** -

2 . 0 + 4 0 (21) 4 . 6 + 4.1 ( 4 8 ) * * 4 15- 3.9 (38) ** 32.2+15.8(100) **

+4.8+

6.6(50)**

2.5+ 3.3(29)

4.1+ 5 8

(41)*

+7.9+

5.7(72)**

2.1+ 3.0(26)

3.8+ 2.7 ( 4 0 ) * *

a Net nuclear grain counts of duplicate autoradmgraphs. Rat values are the mean of data obtained from 3 ammals. Grain counts included cells with no nuclear labelling encountered in the 50 cells counted for each slide. N umbe rs m parentheses are the percent of cells with net nuclear labellmg >/5 grains. * p < 0.01, * * p < 0 001 versus corresponding controls (Student's t-test, 2-tailed).

70

DN.4-damagmg activity and reduction of DN,4 reparr The results of D N A d a m a g e / a l k a l i n e elutlon assays (Table 4) indicate that a minimal but statistically significant increase in D N A elution rate, due to the induction of single-strand breaks a n d / o r alkali-labile sites, was produced in V79 cells by 1-h exposure to 4 m M CAP, a concentration that did not produce immediate cell death. A similar DNA-damaging effect occurred in rat hepatocytes exposed to 2 m M CAP for 20 h, and was slightly increased by G S H depletion produced by the concomitant exposure to 1 m M BSO. Consistent with these findings the primary cultures of rat hepatocytes treated for 20 h with 2 m M CAP displayed a very modest but statistically sigmficant amount of DNA-repalr synthesis (Table 5). However, CAP never produced an increase over controls of 5 net nuclear grains, which is usually considered the minimal level for a frankly positive response (Williams, 1977). In identical treatment conditions, CAP produced a greater amount of D N A repair in primary cultures of human hepatocytes obtained from donors 1 and 2; in contrast, the increase over controls in the number of net nuclear grains was lower than 5 in the hepatocytes from donor 3. Neither in rat nor in human hepatocytes did BSO-lnduced G S H depletion modify the genotoxic effect. The constant occurrence of D N A damage and repair in rat and human hepatocytes exposed to the procarcinogen D M N , used as a positive control, demonstrated their metabolic competence. Mutagemc actwtty The data listed in Table 6 indicate that a 1-h exposure to CAP in the absence of a metabolic system produced a modest dose-related increase m the number of T G r clones, statistical significance being reached at the 2 m M concentration. However, the effect did not meet the criterion of mutagenicity adopted by Bradley et al. (1981) who classified a compound as positive in this test if it induced a mutation frequency at least 3 times higher than the spontaneous mutant frequency. When co-cultures of V79 cells and freshly isolated rat hepatocytes were exposed to CAP for 20 h the frequency of T G ~ variants was approximately the

TABLE 6 I N D U C T I O N O F M U T A T I O N TO 6 - T H I O G U A N I N E RESISTANCE IN V79 CELLS Treatment

TG r m u t a n t s / 1 0 6 survivors a H EP -

Control C A P 1 mM C A P 2 mM N M U 1 mM D M N 5 mM

HEP +

16.1+ 3 8 22.9+102 (81-96%) b 35 1_+11.6 * (78-81%) 125 3 _+27.5 * * (63-75%) -

11.7+ 1.9 127_+ 2.3 (93--98%) 12.7_+ 3.6 (79-95%) 1302_+447 **

(83-88%) a Values are the means + SD of data obtained from 3 independent experiments. The duration of exposure to the test compound was 1 h in the absence and 20 h in the presence of co-cultured rat hepatocytes. b Numbers m parentheses indicate relative survival range * p < 0.05, * * p < 0.01 vs. controls (significance level deterrmned according to K a s t e nba um and Bowman, 1970).

same as that displayed by controls. Both N M U (1 mM) and D M N (5 mM), used as positive controls in the 2 experimental conditions, yielded a high frequency of T G r mutants at approximately equitoxic doses.

Mwronucleus test Administration to rats by the oral route of a single dose of 1250 m g / k g CAP, which corresponds to ½ LDs0, failed to induce an increased incidence of both micronucleated hepatocytes and polychromatic erythrocytes (Table 7). The ratio of normochromatic to polychromatic erythrocytes TA B LE 7 F R E Q U E N C Y O F M I C R O N U C L E A T E D CELLS IN RATS TREATED WITH CAP Treatment

Control C A P 1250 m g / k g p.o. D M N 10 m g / k g Lp.

Frequency of n'ncronucleated cells ( × 1 0 - 3) (mean + SD) Polychromatlc erythrocytes

Hepatocytes

2.78 + 2.28 1.99 + 0.99 9.60 + 5 46 *

3.58 + 0.85 3.57 + 0.62 12.70 + 2 78 *

p < 0.001 VS corresponding controls (Student's t-test, 2tatled).

71

was in the range of normal values. D M N produced micronucleated cells in both tissues.

Discussion The results of the present study, undertaken to explore the possible genotoxic effect of CAP on mammalian cells, indicate that this antimicrobial agent causes very weak positive responses in in vitro systems at concentrations (2-4 mM) markedly higher than those (0.08-0.15 mM) occurring after exposure to maximal therapeutic doses. In brief, a minimal amount of D N A fragmentation was detected in both V79 cells and rat hepatocytes, and the occurrence of DNA lesions was confirmed by a modest increase in the frequency of T G r mutants in the former and of marginal DNA-repair synthesis in the latter cell type. The low genotoxicity of CAP might be related to the limiting effect of its cytotoxicity and to its capability to act as an inhibitor of cytochrome P-450 isoenzyme activity. The amount of DNA-repair synthesis reached the level judged indicative of a positive response in the hepatocyte cultures from 2 of 3 human donors, and this might be a sign of a greater susceptibility in some individuals of our species. In contrast, in vivo experiments demonstrated that CAP does not produce an increase in the frequency of micronucleated cells in the liver and in the bone marrow of intact rats. Since CAP is a nitroaromatic compound, its DNA-damaging activity should be attributed, as previously proposed by Rosenkranz (1988) and Yunis (1988), to the sequential reduction of the nitro group to nltroso and N-hydroxylamlno reactive derivatives. As a matter of fact both the cytosol and the microsome of rat liver have been shown to reduce CAP to the corresponding amine (Fouts and Brodie, 1956), and damage of DNA has been detected during electrochemical reduction of CAP (Skolimowskl et al., 1981). Our resuits do not support a nitroreductive mechanism of CAP genotoxicity. In fact, DNA fragmentation was of a similar mimmal degree in V79 cells and in rat hepatocytes, while a greater nitroreductive activity has to be expected in liver cells; the mutagenic activity of CAP for V79 cells was abolished by their co-culture with rat hepatocytes; in spite

of the fact that the interaction of aromatic nitroso groups with GSH is known to result in their inactivation (Eyer and Schneller, 1983), the BSOinduced GSH depletion produced only a marginal increase in D N A damage and did not influence the amount of DNA repair in either rat or human hepatocytes. On the other hand, aplastic anaemia, a devastating complication of CAP therapy probably associated with an increased risk of leukaemaa, has been related to a particular sensitivity to CAP metabolites. The evidence of a very low hepatic activation to reactive species of this antimicrobial agent, supported by our results, strengthens the hypothesis, already suggested by Yunis (1988), that in vivo CAP metabolism is principally catalysed by gut mlcroflora. This hypothesis might also explain the very limited evidence of genotoxic effects in rodents, where the intestinal metabolism could be different from that occurring in humans. In conclusion, we deem that the recent statement of IARC (1989) that CAP is probably carcinogenic to humans should be taken into due account, and in this respect it is worth noting that the large number of broad-spectrum antibacterial agents presently available undoubtedly allows a reduction of CAP indications.

Acknowledgements This work was supported by the Consiglio Nazionale delle Ricerche, Finalized Project 'Oncology' (Contract 88.00545.44), and by funds of the Public Instruction Ministry.

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