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Differential DNA adduct formation and disappearance in three mouse tissues after treatment with the mycotoxin ochratoxin A
Mutation Research, 289 (1993) 265-273 © 1993 Elsevier Science Publishers B.V. All rights reserved 0027-5107/93/$06.00
265
MUT 05283
Differential D N A adduct formation and disappearance in three mouse tissues after t r e a t m e n t with the mycotoxin ochratoxin A Annie Pfohl-Leszkowicz
a
Yann Grosse
a
Amadou Kane a,b Edmond E. Creppy c
and Guy Dirheimer
a
Institut de Biologie Mol&ulaire et Cellulaire du Centre National de la Recherche Scientifique and Universitd Louis Pasteur, Strasbourg, France, b Institut de Technologie Alimentaire, Dakar, Sdn~gal and c Laboratoire de Toxicologie et d'HygiOne Appliqude, Unicersitd Bordeaux II, Bordeaux, France (Received 18 February 1993) (Revision received 25 May 1993) (Accepted 1 June 1993)
Keywords: Ochratoxin A; DNA adducts; Genotoxicity
Summary Ochratoxin A (OTA) is a mycotoxin which has been implicated in Balkan endemic nephropathy, a disease characterized by a high incidence of urinary tract tumors. It induces DNA single-strand breaks and has been shown to be carcinogenic in two rodent species. For a better understanding of the OTA genotoxic effect, OTA-DNA adduct formation and disappearance has been measured using the 32p-postlabelling method after oral administration of 2.5 mg/kg of OTA to mice. In kidney, liver and spleen, several modified nucleotides were clearly detected in DNA, 24 h after administration of OTA, but their level varied significantly in a tissue and time dependent manner over a 16-day period. Total DNA adducts reached a maximum at 48 h when 103, 42 and 2.2 adducts per 1 0 9 nucleotides were found respectively in kidney, liver and spleen, indicating that kidney is the main target of the genotoxicity and likely carcinogenicity of OTA. The major adduct differed between kidney and liver. All adducts disappeared in liver and spleen 5 days after compound administration, whereas some adducts persisted for at least 16 days in the kidney. Some adducts were organ specific. The finding that the adducts are not quantitatively and qualitatively the same in the three organs examined is likely due to differences of metabolism in these organs, leading to different ultimate carcinogens and may also result from differences in the efficiency of repair processes.
Ochratoxin A (OTA) is a naturally occurring mycotoxin produced by several species of the fungal genera Aspergillus and Penicillium. Its Correspondence: Dr. A. Pfohl-Leszkowicz, Institut de Biologie Mol&ulaire et Cellulaire du Centre National de la Recherche Scientifique and Universit6 Louis Pasteur, 15, rue Descartes, 67084 Strasbourg, France.
occurrence in plant and animal products has been well documented (Krogh, 1987). It consists of a chlorinated dihydroisocoumarin moiety linked through its 7-carboxyl group by an amide bond to L-/3-phenylalanine (Fig. 1). OTA has been shown to have a number of toxic effects, the most prominent being nephrotoxicity (Krogh, 1992). Similarities between OTA-induced porcine nephropathy
266
CI Fig. 1. Structure of ochratoxin A.
and human Balkan endemic nephropathy have been noted (Krogh et al., 1977). Furthermore, OTA has immunosuppressive and teratogenic effects (for a recent review see Pohland et al., 1992). Dietary feeding of ochratoxin A induced renal adenomas and hepatocellular carcinomas in mice (Kanizawa and Suzuki, 1978; Kanizawa, 1984; Bendele et al., 1985a) and in rats (Boorman, 1989). Carcinogenicity of OTA to humans is suspected because of the high incidence of kidney, pelvis, ureter and urinary bladder carcinomas among patients suffering from Balkan endemic nephropathy (Castegnaro et al., 1987; Ceovi6 et al., 1992; Vukeli6 et al., 1992). In regions with Balkan endemic nephropathy, high levels of ochratoxin A were found in human blood (Petkova-Bocharova and Castegnaro, 1991). However, the mycotoxin has also been found in other European countries, Czechoslovakia, Germany, Poland and Sweden (for a review see Hald, 1992) and more recently in France (Creppy et al., 1991). The genotoxic status of OTA was controversial since almost all microbial and mammalian assays were negative (Engel and von Milczewski, 1976; Ueno and Kubota, 1976; Umeda et al., 1977; Kuczuk et al., 1978; Wehner et al., 1978; Bartsch et al., 1980). A weakly positive response for induction of unscheduled DNA synthesis in primary hepatocytes from ACI C3H strain mice and ACI strain rats was found by Mori et al. (1984) but not by Bendele et al. (1985). Contradictory results were also found for sister-chromatid exchange in CHO cells (for a review, see KuiperGoodman and Scott, 1989). Recently, Manolova et al. (1990) demonstrated chromosomal aberrations, particularly on X chromosomes, produced in human lymphocytes in culture by ochratoxin A at a concentration of 15 nM. Moreover, Creppy et al. (1985) detected DNA damage reflected by
single-strand breaks in kidney, liver and spleen of male BALB/c mice injected intraperitoneally with OTA. The DNA damage to splenic cells was confirmed in vitro. DNA single-strand breaks have also been reported in renal and hepatic tissues of rats subchronically given small amounts of OTA by gavage (Kane et al., 1986). Recently, Malaveille et al. (1991) have shown that OTA induces SOSDNA repair directly in E. coli PQ37 strain. In addition, a rat hepatocyte mediated mutagenic response was demonstrated in Salmonella typhimurium TA1535, 1538 and 100 strains by Hennig et al. (1991). To better understand the mechanism of OTA carcinogenesis, we searched for OTA-DNA adducts using the nuclease P1 enrichment version of the 32p-postlabelling method (Reddy and Randerath, 1986). In a preliminary study, several adducts were found in three organs of mice treated with three doses of ochratoxin A (PfohlLeszkowicz et al., 1991). The DNA adduct level was dose dependent and time related. Since most of the adducts disappeared within 3 days, after administration of 0.6 or 1.2 mg/kg body weight, the study reported here was focused on DNA adduct levels after administation of 2.5 mg/kg of OTA determined over a 16-day period. Materials and methods
Chemicals Ochratoxin A, proteinase K, apyrase and ribonucleases A and T1 were obtained from Sigma (St. Louis, MO, USA), T4 polynucleotide kinase from PL Biochemicals (Milwaukee, Wl, USA), micrococcal nuclease and spleen phosphodiesterase from Worthington Biochemicals (Freehold, N J, USA), nuclease P1 from Boehringer (Mannheim, Germany) and [y32p]ATP, 5000 Ci/mmole from Amersham (Buckinghamshire, UK). Treatment of animals Swiss male mice (Sexal, Vigneul sous Montm6dy, France) weighing 25 + 2 g, aged 7 weeks, were given OTA 2.5 mg/kg body weight in 0.1 M NaHCO 3 pH 7.4 by gastric intubation. They were killed by decapitation after 8, 24, 48, 72, 120, 192 or 384 h. Two animals were used for each time
267
point. Kidney, liver and spleen were excised and frozen at -80°C until further processing.
32p-Postlabelling of DNA adducts The DNAs were extracted and purified as described previously (Pfohl-Leszkowicz et al., 1991). The method used for 32p-postlabelling was that previously described by Reddy and Randerath (1986) with minor modifications. Briefly, DNA (6 ~zg) was digested at 37°C for 4 h with micrococcal nuclease (183 mU) and spleen phosphodiesterase (12 mU) in a reaction mixture (total volume 10 /zl) containing 20 mM sodium succinate and 10 mM CaC12, pH 6. In a separate experiment, it was checked that no dinucleotides remained after this treatment. Digested DNA was treated with nuclease P1 (6 /zg) at 37°C for 45 min before 32p-postlabelling according to Reddy and Randerath (1986). Normal nucleotides and pyrophosphate were removed by chromatography on polyethyleneimine-celluloseplates in 2.3 M NaH2PO 4 pH 5.7 (D1) overnight. Origin areas containing labelled adducted nucleotides were cut out and transferred on to another polyethyleneimine-cellulose plate, which was run in 4.77 M lithium formate and 7.65 M urea pH 3.5 for 4.5 h (D2). Two further migrations (D3 and D4) were performed perpendicularly to D2.
The solvent for D3 was 0.6 M NaH2PO 4 and 5.95 M urea pH 6 for 3 h, and the solvent for D4 was 1.7 M NaH2PO 4 pH 6 for 2 h. Autoradiography was carried out at -80°C for 24 or 48 h in the presence of an intensifying screen (Cronex). Spots were scraped off and their radioactivity counted by the Cerenkov technique. Results
The total amount of kidney, liver and spleen DNA adducts measured as a function of time is shown in Fig. 2. Adducts were 2-fold lower in liver and 19-fold lower in spleen than in kidney at 24 h. These values represent 40.6, 19.4 and 2.1 adducts per 10 9 nucleotides for kidney, liver and spleen respectively. They reached a maximum at 48 h: 103, 42 and 2.2 adducts per 10 9 nucleotides in kidney, liver and spleen respectively. In spleen, almost all adducts were repaired within 72 h, whereas in liver, they were repaired within 8 days. In kidney 23.3 and 7.7 adducts per 10 9 nucleotides were still detected 8 days and 16 days respectively after OTA administration, indicating that the kidney may also be the main target for the genotoxicity of OTA, as it is for nephrotoxicity.
120
100
80
~
60
-~
40
,L
20
1
~
8h
24h
I
I
48h
72h
,1 ,11 120h
192h
-q
384h
Time of exposure
Fig. 2. Total D N A adduct levels in kidney, liver and spleen after treatment with 2.5 m g / k g of ochratoxin A. Results are expressed as number of adduct per 10 9 nucleotides ( = relative adduct level)
268 The pattern of the adducts in kidney is shown in Fig. 3. Quantitative estimates of the different adducts are given in Table 1. In kidney, 24 adducts were detected. Four of them (Nos. 1, 5, 18 and 20) appeared as early as 8 h and were very persistent. Twelve of them (Nos. 1, 2, 5, 7, 8, 13, 17, 18, 20, 24, 30 and 33) were present at 24 h. Ten (Nos. 3, 4, 9, 11, 12, 14, 15, 19, 21, 22) appeared only at 48 h. Adduct No. 32 appeared only at 72 h, increased at 120 h but
disappeared at 192 h. Two adducts (Nos. 13 and 30) reached their maximum level at 24 h; thirteen (Nos. 2, 4, 7, 8, 9, 11, 12, 14, 17, 20, 21, 24, 33) reached their maximum level at 48 h, four at 72 h (Nos. 1, 3, 5 and 18) and four at 120 h (Nos. 18, 19, 22, 24, 32). Adduct No. 1 was dominant and represented 36% of the total at 72 h. Adduct No. 33 represented 16% of the total at 48 h, however it decreased more rapidly than adduct No. 1. Some adducts were very persistent, seven of them
A Cent~ Kidney
J 22
13~
12 7
33
I~M
.
.
.
.
.
192H
Fig. 3. (A) Autoradiograms of mouse kidney DNA adducts after administration per os of a single dose of 2.5 mg/kg of ochratoxin A. (B) Numbering of the adducts.
269
B,
19
22
O
,o P "
.Ooo
nant one and the most persistent in kidney, disappeared by 72 h in liver DNA. The major adduct in liver at 24 h was adduct No. 5. It represented 26% of the total. At 48 h, adducts 8 and 10 were the major ones and represented respectively 15 and 14% of the total. Five adducts disappeared at 72 h (Nos. 1, 10, 11, 13, and 30); six others disappeared at 120 h (Nos. 5, 7, 9, 15, 18 and x). After 192 h, all disappeared including adducts y and z which appeared only at 72 h. In spleen, there were fewer adducts. Only four (Nos. 1, 12, 20, tr) were detected at 24 h (Fig. 5). Three of them were similar to the kidney DNA adducts. The fourth one (adduct tr) was specific to spleen. Adduct No. 1 was again by far the dominant one in the spleen, representing about 50% of the adducts. It appeared rapidly and persisted up to 120 h. It disappeared at 192 h.
21
O
o
33°
L Or
Fig. 3 (continued).
were still detectable 16 days after a single dose of
OTA (Nos. 1, 5, 7, 18, 20, 22, 33). In liver, only 15 adducts were detected (Fig. 4). From their migration characteristics, 11 may correspond with DNA adducts observed in kidney. Six of them appeared at 24 h in both kidney and liver. Four of them (Nos. 10, x, y, z) were specific to liver DNA. Three of them (Nos. 1, 5, 13) appeared rapidly as they reached their highest level at 24 h. All the others peaked at 48 h except adducts 18, y and z which reached their highest level at 72 h. Adduct No. 1, which was the domi-
Discussion A preliminary study (Pfohl-Leszkowicz et al., 1991) had shown that a single dose of ochratoxin A, a ubiquitous mycotoxin largely found in human blood, causes DNA adduct formation in mouse organs. This study confirms these results although the pattern obtained in the preliminary
TABLE 1 QUANTIFICATION OF KIDNEY, LIVER AND SPLEEN DNA ADDUCTS OF MICE TREATED WITH A SINGLE DOSE OF OCHRATOXIN A Results are expressed as number of adducts per 10 9 nucleotides. Adduct number KIDNEY 8h 24h 48h 72h 120h 192h 38411 LIVER 24h 48h 72h 120h 192h SPLEEN 24 h ,48h 72h 120h 192h
1
2
3
4
5
7
8
9
10
11
12
13
14
15
17
18
19
20
21
22
24
30
32
33
3,0 16.1 21.1 30.8 19.0 9.8 4.5
2.9 9.9 2.0 0.8 0 0
0 1.3 4.7
0 4.9 4.0 0 0 0
0.8 2.9 4.0 4.7 2.6 1.3 0,7
2.8 5.3 2.5 1.8 1,6 0,5
3.6 4.2 2.5 1.4 0 0
0 3.2 2.0 1.2 0 0
0 0 0 0 0 0
0 6.9 1.8 0.5 0 0
0 6.1 3.8 1.8 0.5 0
2.7 2.2 2.0 1.2 1.1 0
0 2,6 1.1 0 0 0
0 0.7 1.6 1.9 0.5 0
1.2 3.6 2.7 2.5 1.4 0
0.6 1.2 2.0 3.3 2.5 1.4 1
0 0,8 1,2 2,2 1.6 0
1.0 2.2 4.0 2,7 1.9 0.9 0.3
0 1.7 0.9 0 0 0
0 0.2 1.5 3.2 1.2 0,3
1.2 2.5 2.0 1,5 0,8 0
3.3 0 0 0 0 0
0 0 0.4 3 0 0
0.6 16.0 7.2 4.3 0.8 0.4
5.1 2.6 1.0 0 0
0.5 1.0 0.5 0 0
2.8 6.6 2.4 1.9 0
0 3.6 1.1 0 0
1.0 5.9 0 0 0
0.7 1.1 0 0 0
0 1.9 0.8 0.6 0
1.5 1.1 0 0 0
3.4 1.0 0 0 0 1.1 1.2 0.5 0,1 0
0.4 0
0.7 0.4 0 0 0
1,7 3,7 0,9 0 0
0 1.0 1.3 0 0
2.5 7.0 0 0 0 0.1 0.5 0 0 0
x
y
z
a
Total
5.4 40.6 103.1 85,6 53.3 23.3 7.7 0.2 4.8 0.4 0 0
0 0 0.6 0.3 0
0 0 1.8 0,9 0
19.4 42.3 10.9 3.7 0 0.2 0.1 0 0 0
2.1 2.2 0.5 0.1 0
270 study was not exactly the same as reported here. The differences observed can be accounted for by modifications which were done to optimize separation. One change was the addition of proteinase K during D N A extraction to remove remaining proteins. This removal of protein revealed spot 33 (Fig. 2) which was previously obscured in the trail of adducts spreading from the origin of the chromatogram. Other alterations include (i) an increase in ionic strength (D2 and D3) as compared to the previous study; (ii) a
decrease in the migration time for D3 to 3 h (instead of 4.5 h); (iii) and a decrease in the size of the W h a t m a n p a p e r from 4 cm to 2 cm. These modifications lead to a 12-fold increase in recovery of the adducts at 48 h and to a 2-fold increase at 72 h, because several adducts (1, 2, 20, 21 and 32) were lost in the W h a t m a n p a p e r in the previous study. This explains why the maximum formation of adducts is now at 48 h instead of 72 h. No D N A adduct was found in control animals, indicating that there was no interference by other
Fig. 4. Autoradiograms of mouse liver DNA adducts (same legend as in Fig. 3).
271
6
1
Fig. 5. Autoradiogram of mouse spleen DNA adducts at 48 h.
genotoxic substances through feed intake or other mechanisms. In contrast, Randerath et al. (1986, 1988) detected DNA adducts, referred to as I spots, in control rats not treated with carcinogens. These compounds were presumably derived from reactive electrophilic by-products of normal metabolic activities. Our results show that these compounds are not observable at the level of one adduct per 101° nucleotides, which is the detection limit of the method of Reddy and Randerath (1986). Thus, low levels of DNA adducts may be less readily detected in the mouse than the rat. Our results also confirmed the genotoxicity of OTA, suggested previously by the observation that it caused single-strand breaks both in vitro and in vivo (Creppy et al., 1985; Kane et al., 1986). The kinetics of appearance of single-strand breaks were parallel to the kinetics of formation of DNA adducts. DNA single-strand breaks and some DNA adducts were detected in kidney 8 h after administration of OTA. In liver, the majority of DNA adducts appeared 24 h after the administration as was the case for DNA singlestrand breaks. However, there was no strict parallelism between DNA single-strand breaks and disappearance of DNA adducts. The two techniques do not monitor the same phenomena and do not have the same sensitivity. DNA single-
strand breaks correspond both to the removal of DNA adducts by endonucleases and to the direct action of free radicals, produced during metabolism, which then create both single- and doublestrand breaks in DNA (Bohr, 1991). The postlabelling method detects bulky adducts covalently bound to DNA and the level of adducts found at a particular sampling time is the net result of formation and repair of these adducts. Thus, the detected adducts correspond to those which are not rapidly repaired. This may mean that the lower level of DNA adducts in liver was due to more efficient repair, whereas in kidney this repair is less rapid. Differential rates of repair could explain why there was an accumulation of adducts in kidney, indicating that kidney is a target organ for carcinogenicity. This was clearly found with the major adduct No. 1 which had already disappeared from liver after 72 h whereas in kidney it culminated at 72 h. It is also true with the other major adducts, Nos. 5 and 12 for example. Some of the adducts, which are not at all found in liver DNA, whereas they appear in kidney, may be very rapidly repaired in liver. Another explanation for the lower number of adducts in liver, as compared with kidney, could come from differences in metabolism of OTA between these organs. Most of the carcinogens giving DNA adducts require metabolism to electrophilic compounds, the so-called ultimate carcinogens (Miller and Miller, 1969). The many different adducts that we found are consistent with OTA being metabolized by several metabolic pathways a n d / o r by formation of adducts on more than one base of the DNA. Such differences in pathways between organs could result in the observed organ-specific adducts. Several metabolites of ochratoxin A from mice, rat and rabbit have been identified. (For reviews see Delacruz and Bach, 1990; Stormer, 1992.) These are hydroxylated compounds [4R]- and [4S]-hydroxyochratoxin A, 10-hydroxyochratoxin A (Stermer et al., 1981, 1983), tyrosine-ochratoxin A (Creppy et al., 1990) and the dihydroisocoumarin moiety of OTA, called OTa, obtained by the cleavage of the peptide bond of the mycotoxin. These metabolites are formed both in vitro and in vivo (StOrmer et al., 1981; Stein et al., 1985; Ueno, 1985). In a pig hepatic system, Oster
272 et al. (1991) have also shown the p r e s e n c e of an O T A derivative which was m o r e lipophilic t h a n the p a r e n t c o m p o u n d . T h e rate of p r o d u c t i o n a n d the n a t u r e of these m e t a b o l i t e s vary from o n e species to a n o t h e r ( S t c r m e r et al., 1981). T o date, for example, 10-hydroxy-OTA has only b e e n f o u n d in rabbit. O t h e r m e t a b o l i t e s of O T A could also be c a n d i d a t e s for causing genotoxicity, m u t a genicity a n d carcinogenicity. T h e s e are possibly the c o n j u g a t e d c o m p o u n d s of O T A , with glutathione, g l u c u r o n i c acid, sulfate, a m i n o acids, etc., or the m e t a b o l i t e s of O T a . In addition, some adducts might be modified on the D N A . This is not a c o m m o n o c c u r r e n c e a n d it could explain the parallel decrease a n d increase of some of the adducts. Structural e l u c i d a t i o n of the O T A adducts will not be easy d u e to the small a m o u n t s of available material. O u r results d e m o n s t r a t e d clear organ-specific effects in the covalent b i n d i n g of O T A - d e r i v e d material to D N A . T h e s e processes, which may have i n c l u d e d differences in metabolic activation pathways a n d variations in the efficiency of D N A repair, lead to different major a d d u c t species in the o r g a n examined. T h e m a j o r a d d u c t of kidney a n d spleen was No. 1, b u t No. 8 for liver. M o r e investigations in vitro a n d in vivo are n e e d e d to d e t e r m i n e how O T A m e t a b o l i s m in different tissues leads to different D N A adducts, the n a t u r e of the modified bases a n d the n a t u r e of the c o m p o u n d s b o u n d to the D N A .
Acknowledgements This research was s u p p o r t e d by grants from the Ligue N a t i o n a l e contre le Cancer, ComitEs D E p a r t e m e n t a u x de la G i r o n d e et d u H a u t - R h i n , the Minist~re de la R e c h e r c h e et de la T e c h n o l o gie ( A c t i o n Toxicologie), the F o n d a t i o n p o u r la R e c h e r c h e MEdicale a n d the REgion A q u i t a i n e , University of B o r d e a u x II. W e also t h a n k Dr. N. M a r t i n for the correction of the English.
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Pfohl-Leszkowicz, A., K. Chakor, E.E. Creppy and G. Dirheimer (1991) DNA-adduct formation in mice treated with ochratoxin A, in: M. Castegnaro, R. Plegtina, G. Dirheimer, I.V. Chernozemsky and H. Bartsch (Eds.), Mycotoxin, Endemic Nephropathy and Urinary Tract Tumours, IARC Sci. Publ. No. 115), International Agency for Research on Cancer, Lyon, pp. 245-253. Pohland, A.E., S. Nesheim and L. Friedman (1992) Ochratoxin A: a review, Pure Appl. Chem., 64, 1029-1046. Randerath, K., M.V. Reddy and R.M. Disher (1986) Age-and tissue-related DNA modifications in untreated rats: detection by 32p-postlabeling assay and possible significance for spontaneous tumor induction and aging, Carcinogenesis, 7, 1615-1617. Randerath, K., L.-J.W. Lu and D. Li (1988) A comparison between different types of covalent DNA modifications (1-compounds, persistent carcinogen adducts and 5-methylcytosine) in regenerating rat liver, Carcinogenesis, 9, 1843-1848. Reddy, M.V. and K. Randerath (1986) Nuclease P1 mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA-adducts, Carcinogenesis, 7, 1543-1551. Stein, A.F., T.D. Phillips, L.F. Kubena and R.B. Harvey (1985) Renal tubular secretion and reabsorption as factors in ochratoxicosis. Effects of probenecid on nephrotoxicity, J. Toxicol. Environ. Health, 16, 593-605. Stormer, F.C. (1992) Ochratoxin A. A mycotoxin of concern, in: D. Bhatnagar, E.B. Lillehoj and D.K. Arora (Eds.), Handbook of Applied Mycology, Dekker, New York, pp. 403-432. St0rmer, F.C., C.H. Hansen, J.l. Pedersen, G. Hvistendahl and A.J. Aasen (1981) Formation of (4R)- and (4S)-4-hydroxyochratoxin A from ochratoxin A by liver microsomes from various species, Appl. Environ. Microbiol., 39, 971975. St0rmer, F.C., O. St6ren, C.H. Hansen, J.I. Pedersen and A.J. Aasen (1983) Formation of (4R)- and (4S)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A from ochratoxin A by rabbit liver microsomes, Appl. Environ. Microbiol., 45, 1183-1187. Ueno, Y. (1985) The toxicology of mycotoxins, CRC Crit. Rev. Toxicol., 14, 99-132. Ueno, Y. and K. Kubota (1976) DNA-attacking ability of carcinogenic mycotoxins in recombination-deficient mutant cells of Bacillus subtilis, Cancer Res., 36, 445-451. Umeda, T.M., T. Tsutsui, M. Ithoh and M. Saito (1977) Mutagenicity and inducibility of DNA single-strand breaks and chromosome aberrations by various mycotoxins, Gann, 68, 619-625. Vukeli6, M., B. So~tari6 and M. Belicza (1992) Pathomorphology of Balkan endemic nephropathy, Food Chem. Toxicol., 30, 193-200. Wehner, F.C., P.G. Thiel, S.J. Van Rensburg and I.P.PC. Demasius (1978) Mutagenicity to Salmonella typhimurium of some Aspergillus and Penicilium toxins, Mutation Res., 58, 193-203.
265
MUT 05283
Differential D N A adduct formation and disappearance in three mouse tissues after t r e a t m e n t with the mycotoxin ochratoxin A Annie Pfohl-Leszkowicz
a
Yann Grosse
a
Amadou Kane a,b Edmond E. Creppy c
and Guy Dirheimer
a
Institut de Biologie Mol&ulaire et Cellulaire du Centre National de la Recherche Scientifique and Universitd Louis Pasteur, Strasbourg, France, b Institut de Technologie Alimentaire, Dakar, Sdn~gal and c Laboratoire de Toxicologie et d'HygiOne Appliqude, Unicersitd Bordeaux II, Bordeaux, France (Received 18 February 1993) (Revision received 25 May 1993) (Accepted 1 June 1993)
Keywords: Ochratoxin A; DNA adducts; Genotoxicity
Summary Ochratoxin A (OTA) is a mycotoxin which has been implicated in Balkan endemic nephropathy, a disease characterized by a high incidence of urinary tract tumors. It induces DNA single-strand breaks and has been shown to be carcinogenic in two rodent species. For a better understanding of the OTA genotoxic effect, OTA-DNA adduct formation and disappearance has been measured using the 32p-postlabelling method after oral administration of 2.5 mg/kg of OTA to mice. In kidney, liver and spleen, several modified nucleotides were clearly detected in DNA, 24 h after administration of OTA, but their level varied significantly in a tissue and time dependent manner over a 16-day period. Total DNA adducts reached a maximum at 48 h when 103, 42 and 2.2 adducts per 1 0 9 nucleotides were found respectively in kidney, liver and spleen, indicating that kidney is the main target of the genotoxicity and likely carcinogenicity of OTA. The major adduct differed between kidney and liver. All adducts disappeared in liver and spleen 5 days after compound administration, whereas some adducts persisted for at least 16 days in the kidney. Some adducts were organ specific. The finding that the adducts are not quantitatively and qualitatively the same in the three organs examined is likely due to differences of metabolism in these organs, leading to different ultimate carcinogens and may also result from differences in the efficiency of repair processes.
Ochratoxin A (OTA) is a naturally occurring mycotoxin produced by several species of the fungal genera Aspergillus and Penicillium. Its Correspondence: Dr. A. Pfohl-Leszkowicz, Institut de Biologie Mol&ulaire et Cellulaire du Centre National de la Recherche Scientifique and Universit6 Louis Pasteur, 15, rue Descartes, 67084 Strasbourg, France.
occurrence in plant and animal products has been well documented (Krogh, 1987). It consists of a chlorinated dihydroisocoumarin moiety linked through its 7-carboxyl group by an amide bond to L-/3-phenylalanine (Fig. 1). OTA has been shown to have a number of toxic effects, the most prominent being nephrotoxicity (Krogh, 1992). Similarities between OTA-induced porcine nephropathy
266
CI Fig. 1. Structure of ochratoxin A.
and human Balkan endemic nephropathy have been noted (Krogh et al., 1977). Furthermore, OTA has immunosuppressive and teratogenic effects (for a recent review see Pohland et al., 1992). Dietary feeding of ochratoxin A induced renal adenomas and hepatocellular carcinomas in mice (Kanizawa and Suzuki, 1978; Kanizawa, 1984; Bendele et al., 1985a) and in rats (Boorman, 1989). Carcinogenicity of OTA to humans is suspected because of the high incidence of kidney, pelvis, ureter and urinary bladder carcinomas among patients suffering from Balkan endemic nephropathy (Castegnaro et al., 1987; Ceovi6 et al., 1992; Vukeli6 et al., 1992). In regions with Balkan endemic nephropathy, high levels of ochratoxin A were found in human blood (Petkova-Bocharova and Castegnaro, 1991). However, the mycotoxin has also been found in other European countries, Czechoslovakia, Germany, Poland and Sweden (for a review see Hald, 1992) and more recently in France (Creppy et al., 1991). The genotoxic status of OTA was controversial since almost all microbial and mammalian assays were negative (Engel and von Milczewski, 1976; Ueno and Kubota, 1976; Umeda et al., 1977; Kuczuk et al., 1978; Wehner et al., 1978; Bartsch et al., 1980). A weakly positive response for induction of unscheduled DNA synthesis in primary hepatocytes from ACI C3H strain mice and ACI strain rats was found by Mori et al. (1984) but not by Bendele et al. (1985). Contradictory results were also found for sister-chromatid exchange in CHO cells (for a review, see KuiperGoodman and Scott, 1989). Recently, Manolova et al. (1990) demonstrated chromosomal aberrations, particularly on X chromosomes, produced in human lymphocytes in culture by ochratoxin A at a concentration of 15 nM. Moreover, Creppy et al. (1985) detected DNA damage reflected by
single-strand breaks in kidney, liver and spleen of male BALB/c mice injected intraperitoneally with OTA. The DNA damage to splenic cells was confirmed in vitro. DNA single-strand breaks have also been reported in renal and hepatic tissues of rats subchronically given small amounts of OTA by gavage (Kane et al., 1986). Recently, Malaveille et al. (1991) have shown that OTA induces SOSDNA repair directly in E. coli PQ37 strain. In addition, a rat hepatocyte mediated mutagenic response was demonstrated in Salmonella typhimurium TA1535, 1538 and 100 strains by Hennig et al. (1991). To better understand the mechanism of OTA carcinogenesis, we searched for OTA-DNA adducts using the nuclease P1 enrichment version of the 32p-postlabelling method (Reddy and Randerath, 1986). In a preliminary study, several adducts were found in three organs of mice treated with three doses of ochratoxin A (PfohlLeszkowicz et al., 1991). The DNA adduct level was dose dependent and time related. Since most of the adducts disappeared within 3 days, after administration of 0.6 or 1.2 mg/kg body weight, the study reported here was focused on DNA adduct levels after administation of 2.5 mg/kg of OTA determined over a 16-day period. Materials and methods
Chemicals Ochratoxin A, proteinase K, apyrase and ribonucleases A and T1 were obtained from Sigma (St. Louis, MO, USA), T4 polynucleotide kinase from PL Biochemicals (Milwaukee, Wl, USA), micrococcal nuclease and spleen phosphodiesterase from Worthington Biochemicals (Freehold, N J, USA), nuclease P1 from Boehringer (Mannheim, Germany) and [y32p]ATP, 5000 Ci/mmole from Amersham (Buckinghamshire, UK). Treatment of animals Swiss male mice (Sexal, Vigneul sous Montm6dy, France) weighing 25 + 2 g, aged 7 weeks, were given OTA 2.5 mg/kg body weight in 0.1 M NaHCO 3 pH 7.4 by gastric intubation. They were killed by decapitation after 8, 24, 48, 72, 120, 192 or 384 h. Two animals were used for each time
267
point. Kidney, liver and spleen were excised and frozen at -80°C until further processing.
32p-Postlabelling of DNA adducts The DNAs were extracted and purified as described previously (Pfohl-Leszkowicz et al., 1991). The method used for 32p-postlabelling was that previously described by Reddy and Randerath (1986) with minor modifications. Briefly, DNA (6 ~zg) was digested at 37°C for 4 h with micrococcal nuclease (183 mU) and spleen phosphodiesterase (12 mU) in a reaction mixture (total volume 10 /zl) containing 20 mM sodium succinate and 10 mM CaC12, pH 6. In a separate experiment, it was checked that no dinucleotides remained after this treatment. Digested DNA was treated with nuclease P1 (6 /zg) at 37°C for 45 min before 32p-postlabelling according to Reddy and Randerath (1986). Normal nucleotides and pyrophosphate were removed by chromatography on polyethyleneimine-celluloseplates in 2.3 M NaH2PO 4 pH 5.7 (D1) overnight. Origin areas containing labelled adducted nucleotides were cut out and transferred on to another polyethyleneimine-cellulose plate, which was run in 4.77 M lithium formate and 7.65 M urea pH 3.5 for 4.5 h (D2). Two further migrations (D3 and D4) were performed perpendicularly to D2.
The solvent for D3 was 0.6 M NaH2PO 4 and 5.95 M urea pH 6 for 3 h, and the solvent for D4 was 1.7 M NaH2PO 4 pH 6 for 2 h. Autoradiography was carried out at -80°C for 24 or 48 h in the presence of an intensifying screen (Cronex). Spots were scraped off and their radioactivity counted by the Cerenkov technique. Results
The total amount of kidney, liver and spleen DNA adducts measured as a function of time is shown in Fig. 2. Adducts were 2-fold lower in liver and 19-fold lower in spleen than in kidney at 24 h. These values represent 40.6, 19.4 and 2.1 adducts per 10 9 nucleotides for kidney, liver and spleen respectively. They reached a maximum at 48 h: 103, 42 and 2.2 adducts per 10 9 nucleotides in kidney, liver and spleen respectively. In spleen, almost all adducts were repaired within 72 h, whereas in liver, they were repaired within 8 days. In kidney 23.3 and 7.7 adducts per 10 9 nucleotides were still detected 8 days and 16 days respectively after OTA administration, indicating that the kidney may also be the main target for the genotoxicity of OTA, as it is for nephrotoxicity.
120
100
80
~
60
-~
40
,L
20
1
~
8h
24h
I
I
48h
72h
,1 ,11 120h
192h
-q
384h
Time of exposure
Fig. 2. Total D N A adduct levels in kidney, liver and spleen after treatment with 2.5 m g / k g of ochratoxin A. Results are expressed as number of adduct per 10 9 nucleotides ( = relative adduct level)
268 The pattern of the adducts in kidney is shown in Fig. 3. Quantitative estimates of the different adducts are given in Table 1. In kidney, 24 adducts were detected. Four of them (Nos. 1, 5, 18 and 20) appeared as early as 8 h and were very persistent. Twelve of them (Nos. 1, 2, 5, 7, 8, 13, 17, 18, 20, 24, 30 and 33) were present at 24 h. Ten (Nos. 3, 4, 9, 11, 12, 14, 15, 19, 21, 22) appeared only at 48 h. Adduct No. 32 appeared only at 72 h, increased at 120 h but
disappeared at 192 h. Two adducts (Nos. 13 and 30) reached their maximum level at 24 h; thirteen (Nos. 2, 4, 7, 8, 9, 11, 12, 14, 17, 20, 21, 24, 33) reached their maximum level at 48 h, four at 72 h (Nos. 1, 3, 5 and 18) and four at 120 h (Nos. 18, 19, 22, 24, 32). Adduct No. 1 was dominant and represented 36% of the total at 72 h. Adduct No. 33 represented 16% of the total at 48 h, however it decreased more rapidly than adduct No. 1. Some adducts were very persistent, seven of them
A Cent~ Kidney
J 22
13~
12 7
33
I~M
.
.
.
.
.
192H
Fig. 3. (A) Autoradiograms of mouse kidney DNA adducts after administration per os of a single dose of 2.5 mg/kg of ochratoxin A. (B) Numbering of the adducts.
269
B,
19
22
O
,o P "
.Ooo
nant one and the most persistent in kidney, disappeared by 72 h in liver DNA. The major adduct in liver at 24 h was adduct No. 5. It represented 26% of the total. At 48 h, adducts 8 and 10 were the major ones and represented respectively 15 and 14% of the total. Five adducts disappeared at 72 h (Nos. 1, 10, 11, 13, and 30); six others disappeared at 120 h (Nos. 5, 7, 9, 15, 18 and x). After 192 h, all disappeared including adducts y and z which appeared only at 72 h. In spleen, there were fewer adducts. Only four (Nos. 1, 12, 20, tr) were detected at 24 h (Fig. 5). Three of them were similar to the kidney DNA adducts. The fourth one (adduct tr) was specific to spleen. Adduct No. 1 was again by far the dominant one in the spleen, representing about 50% of the adducts. It appeared rapidly and persisted up to 120 h. It disappeared at 192 h.
21
O
o
33°
L Or
Fig. 3 (continued).
were still detectable 16 days after a single dose of
OTA (Nos. 1, 5, 7, 18, 20, 22, 33). In liver, only 15 adducts were detected (Fig. 4). From their migration characteristics, 11 may correspond with DNA adducts observed in kidney. Six of them appeared at 24 h in both kidney and liver. Four of them (Nos. 10, x, y, z) were specific to liver DNA. Three of them (Nos. 1, 5, 13) appeared rapidly as they reached their highest level at 24 h. All the others peaked at 48 h except adducts 18, y and z which reached their highest level at 72 h. Adduct No. 1, which was the domi-
Discussion A preliminary study (Pfohl-Leszkowicz et al., 1991) had shown that a single dose of ochratoxin A, a ubiquitous mycotoxin largely found in human blood, causes DNA adduct formation in mouse organs. This study confirms these results although the pattern obtained in the preliminary
TABLE 1 QUANTIFICATION OF KIDNEY, LIVER AND SPLEEN DNA ADDUCTS OF MICE TREATED WITH A SINGLE DOSE OF OCHRATOXIN A Results are expressed as number of adducts per 10 9 nucleotides. Adduct number KIDNEY 8h 24h 48h 72h 120h 192h 38411 LIVER 24h 48h 72h 120h 192h SPLEEN 24 h ,48h 72h 120h 192h
1
2
3
4
5
7
8
9
10
11
12
13
14
15
17
18
19
20
21
22
24
30
32
33
3,0 16.1 21.1 30.8 19.0 9.8 4.5
2.9 9.9 2.0 0.8 0 0
0 1.3 4.7
0 4.9 4.0 0 0 0
0.8 2.9 4.0 4.7 2.6 1.3 0,7
2.8 5.3 2.5 1.8 1,6 0,5
3.6 4.2 2.5 1.4 0 0
0 3.2 2.0 1.2 0 0
0 0 0 0 0 0
0 6.9 1.8 0.5 0 0
0 6.1 3.8 1.8 0.5 0
2.7 2.2 2.0 1.2 1.1 0
0 2,6 1.1 0 0 0
0 0.7 1.6 1.9 0.5 0
1.2 3.6 2.7 2.5 1.4 0
0.6 1.2 2.0 3.3 2.5 1.4 1
0 0,8 1,2 2,2 1.6 0
1.0 2.2 4.0 2,7 1.9 0.9 0.3
0 1.7 0.9 0 0 0
0 0.2 1.5 3.2 1.2 0,3
1.2 2.5 2.0 1,5 0,8 0
3.3 0 0 0 0 0
0 0 0.4 3 0 0
0.6 16.0 7.2 4.3 0.8 0.4
5.1 2.6 1.0 0 0
0.5 1.0 0.5 0 0
2.8 6.6 2.4 1.9 0
0 3.6 1.1 0 0
1.0 5.9 0 0 0
0.7 1.1 0 0 0
0 1.9 0.8 0.6 0
1.5 1.1 0 0 0
3.4 1.0 0 0 0 1.1 1.2 0.5 0,1 0
0.4 0
0.7 0.4 0 0 0
1,7 3,7 0,9 0 0
0 1.0 1.3 0 0
2.5 7.0 0 0 0 0.1 0.5 0 0 0
x
y
z
a
Total
5.4 40.6 103.1 85,6 53.3 23.3 7.7 0.2 4.8 0.4 0 0
0 0 0.6 0.3 0
0 0 1.8 0,9 0
19.4 42.3 10.9 3.7 0 0.2 0.1 0 0 0
2.1 2.2 0.5 0.1 0
270 study was not exactly the same as reported here. The differences observed can be accounted for by modifications which were done to optimize separation. One change was the addition of proteinase K during D N A extraction to remove remaining proteins. This removal of protein revealed spot 33 (Fig. 2) which was previously obscured in the trail of adducts spreading from the origin of the chromatogram. Other alterations include (i) an increase in ionic strength (D2 and D3) as compared to the previous study; (ii) a
decrease in the migration time for D3 to 3 h (instead of 4.5 h); (iii) and a decrease in the size of the W h a t m a n p a p e r from 4 cm to 2 cm. These modifications lead to a 12-fold increase in recovery of the adducts at 48 h and to a 2-fold increase at 72 h, because several adducts (1, 2, 20, 21 and 32) were lost in the W h a t m a n p a p e r in the previous study. This explains why the maximum formation of adducts is now at 48 h instead of 72 h. No D N A adduct was found in control animals, indicating that there was no interference by other
Fig. 4. Autoradiograms of mouse liver DNA adducts (same legend as in Fig. 3).
271
6
1
Fig. 5. Autoradiogram of mouse spleen DNA adducts at 48 h.
genotoxic substances through feed intake or other mechanisms. In contrast, Randerath et al. (1986, 1988) detected DNA adducts, referred to as I spots, in control rats not treated with carcinogens. These compounds were presumably derived from reactive electrophilic by-products of normal metabolic activities. Our results show that these compounds are not observable at the level of one adduct per 101° nucleotides, which is the detection limit of the method of Reddy and Randerath (1986). Thus, low levels of DNA adducts may be less readily detected in the mouse than the rat. Our results also confirmed the genotoxicity of OTA, suggested previously by the observation that it caused single-strand breaks both in vitro and in vivo (Creppy et al., 1985; Kane et al., 1986). The kinetics of appearance of single-strand breaks were parallel to the kinetics of formation of DNA adducts. DNA single-strand breaks and some DNA adducts were detected in kidney 8 h after administration of OTA. In liver, the majority of DNA adducts appeared 24 h after the administration as was the case for DNA singlestrand breaks. However, there was no strict parallelism between DNA single-strand breaks and disappearance of DNA adducts. The two techniques do not monitor the same phenomena and do not have the same sensitivity. DNA single-
strand breaks correspond both to the removal of DNA adducts by endonucleases and to the direct action of free radicals, produced during metabolism, which then create both single- and doublestrand breaks in DNA (Bohr, 1991). The postlabelling method detects bulky adducts covalently bound to DNA and the level of adducts found at a particular sampling time is the net result of formation and repair of these adducts. Thus, the detected adducts correspond to those which are not rapidly repaired. This may mean that the lower level of DNA adducts in liver was due to more efficient repair, whereas in kidney this repair is less rapid. Differential rates of repair could explain why there was an accumulation of adducts in kidney, indicating that kidney is a target organ for carcinogenicity. This was clearly found with the major adduct No. 1 which had already disappeared from liver after 72 h whereas in kidney it culminated at 72 h. It is also true with the other major adducts, Nos. 5 and 12 for example. Some of the adducts, which are not at all found in liver DNA, whereas they appear in kidney, may be very rapidly repaired in liver. Another explanation for the lower number of adducts in liver, as compared with kidney, could come from differences in metabolism of OTA between these organs. Most of the carcinogens giving DNA adducts require metabolism to electrophilic compounds, the so-called ultimate carcinogens (Miller and Miller, 1969). The many different adducts that we found are consistent with OTA being metabolized by several metabolic pathways a n d / o r by formation of adducts on more than one base of the DNA. Such differences in pathways between organs could result in the observed organ-specific adducts. Several metabolites of ochratoxin A from mice, rat and rabbit have been identified. (For reviews see Delacruz and Bach, 1990; Stormer, 1992.) These are hydroxylated compounds [4R]- and [4S]-hydroxyochratoxin A, 10-hydroxyochratoxin A (Stermer et al., 1981, 1983), tyrosine-ochratoxin A (Creppy et al., 1990) and the dihydroisocoumarin moiety of OTA, called OTa, obtained by the cleavage of the peptide bond of the mycotoxin. These metabolites are formed both in vitro and in vivo (StOrmer et al., 1981; Stein et al., 1985; Ueno, 1985). In a pig hepatic system, Oster
272 et al. (1991) have also shown the p r e s e n c e of an O T A derivative which was m o r e lipophilic t h a n the p a r e n t c o m p o u n d . T h e rate of p r o d u c t i o n a n d the n a t u r e of these m e t a b o l i t e s vary from o n e species to a n o t h e r ( S t c r m e r et al., 1981). T o date, for example, 10-hydroxy-OTA has only b e e n f o u n d in rabbit. O t h e r m e t a b o l i t e s of O T A could also be c a n d i d a t e s for causing genotoxicity, m u t a genicity a n d carcinogenicity. T h e s e are possibly the c o n j u g a t e d c o m p o u n d s of O T A , with glutathione, g l u c u r o n i c acid, sulfate, a m i n o acids, etc., or the m e t a b o l i t e s of O T a . In addition, some adducts might be modified on the D N A . This is not a c o m m o n o c c u r r e n c e a n d it could explain the parallel decrease a n d increase of some of the adducts. Structural e l u c i d a t i o n of the O T A adducts will not be easy d u e to the small a m o u n t s of available material. O u r results d e m o n s t r a t e d clear organ-specific effects in the covalent b i n d i n g of O T A - d e r i v e d material to D N A . T h e s e processes, which may have i n c l u d e d differences in metabolic activation pathways a n d variations in the efficiency of D N A repair, lead to different major a d d u c t species in the o r g a n examined. T h e m a j o r a d d u c t of kidney a n d spleen was No. 1, b u t No. 8 for liver. M o r e investigations in vitro a n d in vivo are n e e d e d to d e t e r m i n e how O T A m e t a b o l i s m in different tissues leads to different D N A adducts, the n a t u r e of the modified bases a n d the n a t u r e of the c o m p o u n d s b o u n d to the D N A .
Acknowledgements This research was s u p p o r t e d by grants from the Ligue N a t i o n a l e contre le Cancer, ComitEs D E p a r t e m e n t a u x de la G i r o n d e et d u H a u t - R h i n , the Minist~re de la R e c h e r c h e et de la T e c h n o l o gie ( A c t i o n Toxicologie), the F o n d a t i o n p o u r la R e c h e r c h e MEdicale a n d the REgion A q u i t a i n e , University of B o r d e a u x II. W e also t h a n k Dr. N. M a r t i n for the correction of the English.
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