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In vivo biotransformation of aflatoxin B1 and its interaction with cellular macromolecules in neonatal rats
Mechanisms of Ageing and Development
ELSEVIER
anddWel!lpment
78 (1995) 189-196
In vivo biotransformation of aflatoxin Bl and its interaction with cellular macromolecules in neonatal rats Mohamadreza
Chelcheleh, Abdolamir Allameh*
Department of Biochemistry, School of Medical Sciences, Tarbiat Modaress University, P.O. Box 14155-4838, Tehran, Iran
Received 8 June 1994; revision received 12 October 1994; accepted 11 November 1994
Abstract In this study, the ability of neonatal rat liver to metabolise [3H]aflatoxin Bl (AFBl) was compared to that of the adult animal. In order to make this comparison, neonatal and young adult rats were killed 2, 6, 12 and 24 h after injection with a single i.p. dose of AFBl. The rate of AFBl adduct formation to nuclear DNA and protein was measured in hepatic and pulmonary tissues. The results demonstrated that AFBl was epoxidized more rapidly by the adult’:; liver and lungs 2 h after the toxin administration, compared with those of the neonatal’s (adult 30 pmol and neonatal 12 pmol AFBl bound/mg DNA). However, these differences were more pronounced in hepatic than in pulmonary tissues. The same differences between AFBl-DNA adducts were also observed at different time points. These changes are certainly related to the level of hepatic cytochrome P-450. The delayed cytochrome P-450-dependent AFBl activation in neonatal’s liver provides time enough for de-epoxidation of slowly generated epoxide. The rate of AFBl-epoxide formation at this age was consistent with the activity of phase II metabolism of AFBl (glutathione conjugation). In addition, the hydrolysis of AFBl-DNA adducts at a relatively higher rate by neonatal’s liver may also contribute to the quick removal of the adducts. In spite of the aforementioned evidence which shows the capability of neonatal liver to handle AFBl, the fate of large amounts of free (non-metabolised) AFBl deposited in neonatal’s liver is not well understood.
Keywor&: Aflatoxin Bl; Neonatal;
Biotransformation;
DNA; Protein
* Corresponding author. 0047-t~374/95/$09.50
0 1995 Elsevier Science Ireland Ltd. All rights reserved
SSDZ 0047-6374(94)01536-U
190
M. Chelcheleh, A. Allameh / Mech. Ageing Dee. 78 (1995) 189-196
1. Introduction Aflatoxin Bl (AFBl) is a mycotoxin produced as a secondary metabolite by the toxigenic strains of Aspergillusflavus and A. parasiticus. It is a natural contaminant of the food chain and is a potent hepatocarcinogen in several animal species -[I]. AFBl exerts its biological effects after activation by cytochrome P-450-dependent monooxygenase to its reactive form, AFBl 2,3-epoxide, leading to covalent binding to the cellular macromolecules, particularly DNA [2,3]. In recent years, it has been observed [4] that, like adults, infant humans are exposed to AFBl and congeners. Furthermore, detection of aflatoxins in the cord blood and newborn serum indicated the transfer of these toxins to the neonatal animals. We have already studied the distribution of transmammary AFBl hydroxylmetabolites in neonatal tissues consuming milk from mothers treated with AFBl. In addition, interaction between these metabolites and cellular macromolecules such as DNA, RNA and proteins have been quantitated [5]. This was an approach for the evaluation of the ability of a neonatal rat to handle a chemical carcinogen. However, in this study the mother’s liver is believed to play the central role in the biotransformation of the carcinogen. Therefore, in this study it was difficult to rationalize the part played by the newborn liver in the metabolism of AFBI. A more recent report from our laboratory [6] clearly showed that xenobiotic metabolizing factors are underdeveloped in neonatal’s liver. Microsomal cytochrome P-450-mediated epoxidation of AFBI as measured by its binding to calf thymus DNA in vitro and inhibition of AFBl-DNA binding by cytosolic glutathione (GSH) S-transferase in the reconstitution experiments also showed the low efficiency of neonatal’s liver in metabolising the toxin. The present report is an in vivo comparative study of the AFBl biotransformation in neonatal and adult animals. AFBl adduct formation and removal to the cellular DNA and protein was studied in neonatal rats at different intervals of time to examine the capacity of neonatal tissue in hydrolysing the already formed adducts. The role of the detoxification pathway in controlling the AFBl-DNA binding in neonatal’s liver was evaluated by depleting the GSH level in this tissue prior to carcinogen administration. In addition to this, the possible role of lungs as an extrahepatic organ in sharing the metabolism of AFBI was also investigated.
2. Materials and methods 2.1. Chemicals
[3H]AFB1 was purchased from American Radiolabled Chemical Co., St. Louis, MO. Non-radioactive AFBl, diethyl maleate (DEM), reduced glutathione (GSH), bovine serum albumin, calf thymus DNA, RNA, RNAase, PPO, POPOP, DMSO and DTNB [5,5-di-thio bis-(2-nitrobenzoic acid)] were the products of Sigma Chemical Co., St. Louis, MO. All other reagents and solvents were obtained from E. Merck, Germany.
M. Chelcheleh, A. Allameh / Mech. Ageing Dev. 78 (1995) 189-196
191
2.2. Animals and treatment In this study, albino rats of Wistar strain were selected. Breeding was carried out at the animal house of the School of Medical Sciences at this university. Wherever indicated in the text, adult rats means young adult male rats, 3-5 months old. Neonatal rats were selected on the basis of their birth date and were 17 f 5 days old. All the newborn rats were determined to be male. In each experiment, four young adult rats along with lo-12 neonatal rats were selected for AFBl-treatment. Each rat was administered intraperitoneally (i.p.) with a single dose of [3H]AFB1 (specific activity 18 mCi/mmol) and sacrificed at different time intervals. Tissues from 3-4 newborns were pooled and processed for further studies. In order to compare the pattern of distribution of aflatoxin metabolites in different tissues of adult and neonatal animals, liver, kidneys, lung, intestine and stomach were removed, homogenized and radioactivity was measured. Similarly radioactivity was measured in serum samples obtained from animals prior to sacrifice [ll]. Aliquots of the lung and liver homogenate were taken for the extraction of DNA, RNA and proteins. Extraction and quantitation of AFBI adduct formation to these macromolecules was performed as described earlier [S]. 2.3. Hepatic glutathione depletion by DEM In a set of experiments, the level of cellular hepatic GSH was depleted by i.p. administration of a single dose of DEM (0.6 ml/kg body wt.) 2 h prior to AFBl administration as described above. The level of hepatic GSH was measured using Ellman’s reagent according to Sedlak and Lindsay [7]. 2.4. Other assays Protein levels were measured by Biuret reaction using bovine serum albumin (BSA) as standard [8]. DNA was estimated calorimetrically using diphenylamine reagent based on the Burton method [9]. The amount of RNA associated with the isolated DNA was < 10% as measured by Orcinol reagent according to Disch’s procedure [lo]. 2.5. Statistical analysis Statistical comparison between neonatal and adult animals was performed by non-parametric Wilcoxon and Kruskal-Wallis tests using the SPSS computer program. P-values lower than 0.05 (P < 0.05) were considered significant. 3. Results Ad.ministration of a single i.p. dose of [3H]AFB1 (8 ,uCi [3H]AFB1 containing 40 pg AFBljlOO g body wt.) to neonatal and young adult rats resulted in a differential pattern of distribution of AFBl-metabolites in various tissues [I 11. Hepatic and pulmonary tissues obtained from t3H]AFBl-treated animals were further processed for measurement of AFBl-DNA binding in both the age-groups (Table 1). The data presented in this table illustrate that AFBl adduct formation to DNA and proteins is significantly higher in adult compared to neonate’s liver.
192
M. Chelcheleh, A. Ailameh / Mech. Ageing Del). 78 (1995) 189-196
Table 1 Comparison of AFBl interaction with tissue DNA and protein in adult and neonatal rat: 2 h after AFBl treatment Tissue
Age group
AFBl-macromolecule DNA
Liver Lung
A NN A NN
119 * 34 k 3.9 * 2.9 +
binding Protein
3.4* 2.4 0.4* 0.2
4.8 * 1.1* 1.6kO.6 0.37 + 0.03* 0.27 k 0.03
A, adult; NN, neonatal; results are expressed as mean f S.E.M obtained from four samples from each group; experimental details are as described in ‘Materials and methods’ section; data are in terms of pmol [“H]AFBl bound per mg DNA. *P < 0.05 considered significant.
Low levels of AFBl adduct formation to lung macromolecules were not comparable to that of the liver in both age groups. Table 1 shows that AFBl-protein adduct was always considerably less than the adducts formed to nuclear DNA (adult 4.8 and neonatal 1.6 pmol AFBl bound/mg protein). The level of nuclear RNA isolated from these tissues was extremely low and not sufficient for measuring AFBl-RNA binding. Depletion of GSH by DEM to about 50% in neonatal liver caused an approximately 2.5-fold increase in AFBl-DNA binding. The role of neonatal hepatic glutathione in the AFBl adduct formation to DNA and protein is demonstrated in Table 2. The results of time-course adduct formation and removal of AFBl to nuclear DNA in adult and neonatal rats is compared in Fig. 1. Maximum binding in both the age groups occurred at the same time (6 h after AFBl treatment). The binding of AFBl to the adult liver DNA is not only higher, but it also found to persist longer than in the neonatal tissues (Fig. 1). It is interesting to note that the amount of residual bindings remaining after 24 h of AFBl treatment was considerably lower in the case of neonatal liver (adult 30 and neonatal 12 pmol per mg DNA). Similarly AFBl-protein interaction in adult liver was observed to be higher than that of the neonatal rats (Fig. 2). Table 2 Effect of cellular GSH-depletion on AFBl-macromolecule Treatment
Control DEM
GSH concentration umol/g liver
5.2 k 0.05* 4.2 k 0.09
binding in neonatal rat liver
AFBl-binding DNA
Protein
68.5 + 3.0 167 & 8.8*
1.5 + 0.06 3.0 f 0.09*
Data are expressed as mean k S.E.M of four samples each pooled from 3-4 livers in each group; non protein GSH concentration in liver homogenate was measured according to the Sedlak and Lindsay method [7]. *P < 0.05 considered significant.
M. Chelcheleh, A. Allameh / Mech. Ageing Dev. 78 (1995) 189-196
193
PmoVmg DNA l4CI )
t 1
I
5
10
f I
I
I
16
20
26
hours -
Adult
-+
Newborn
Fig. 1. Kinetics of the persistence of AFBl-DNA adduct in newborn and adult rat liver. Animals receive,d a single i.p. dose of [3H]AFBl and were sacrificed at different time intervals after treatment. AFBl-DNA binding is expressed as pmol [‘HIAFBI bound to milligrams of DNA. Values represent the mean + S.E.M. of three tissue samples.
4. Dixussion
The molecular niechanism(s) of hepatic preneoplastic formation due to AFBl treatment is well understood. Important determinants in the susceptibility of AFBl hepatocarcinogenesis include the enzyme-mediated activation, detoxification and PmoVmg Protein 61
Oi 0
,
I
10
5
I
I
J
16
20
25
hours -
Adult
-J-
Newborn
Fig. 2. Kinetics of [3H]AFBl-protein binding in adult and neonatal rat liver. Experimental details including animal treatments are as described in Fig. 1 legend. Values represent the mean + S.E.M. of three samples.
194
M. Chelcheleh,
A. Allameh / Mech. Ageing Dev. 78 (1995) 189-196
AFBl-DNA adduct formation and removal (repair) [12- 141. There is a good correlation between the level of covalent AFBl-DNA binding in liver slices with the susceptibility of AFBl-induced carcinogenesis in vivo in various animal species [15-171. Accordingly, laboratory animals have been classified as susceptible (rat) and resistant (hamster and mice) species based on their relative extent of biotransformation of AFB 1 [ 18-201. The susceptibility varies greatly between the different species, however within species susceptibility differs with regard to sex, age and nutritional status. The extent of the influence of age on the AFBl-induced hepatocarcinogenesis is not well understood. The presence of some important determinants of susceptibility in neonatal rats such as the difference in the rate of AFBl-DNA adducts in neonatal and adult organs in vivo indicates that the metabolic activation of AFBl is less efficient in neonatal liver. The in vitro microsome-mediated binding experiments clearly showed that, in neonatal liver as in adults, cytochrome P-450 is mainly responsible for the metabolic activation of AFBl. It has been well established that epoxidation alone cannot account for differences in AFBl-DNA binding, since the cytosolic GSH S-transferase plays a major role in the modulation of hepatic AFBl-DNA adduct formation [18,21251. Therefore, due to the reduced level of GSH and GSH S-transferase activity in neonatal liver, the rate of AFBl-GSH formation is also low at this age. Correlation between the depletion of the cellular GSH content in neonatal liver and the increased level of AFBl adduct formation to the nuclear DNA and proteins (Table 2), further substantiates the role of the GSH conjugation system in scavenging the AFBl-epoxide formed in vivo. Because of this close association between the two phases of the metabolism, it is likely that, though often below normal levels, the activity of the GSH system appears sufficient for the small amounts of the epoxide formed in neonatal liver during the phase I metabolism. An important factor which contributes to the susceptibility of neonatal rat is the deposition of large amounts of free AFBl (non-metabolised) in liver at this age. It means that the carcinogen is partially metabolised in immature liver. Hence the complete metabolism of AFBl by neonatal tissue relies on the age-dependent development of the key factors involved in the biotransformation of AFBl. These results raise the question whether alternative mechanisms are present by which the damage to cellular macromolecules caused by AFBl in neonatal rat could be prevented or delayed. The results of time-course AFBl adduct formation and removal to the liver nuclear DNA presented in Fig. 1 reveal that the main reason for the reduced AFBl-DNA interaction is the cytochrome P-450 content in the neonatal tissue. The difference in the catalytic activity of the enzyme in neonatal and adult animals in the presence of AFBl remains almost constant at different time intervals. The adducts measured 24 h after AFBl administration which are considered as residual bindings are believed to be responsible for AFB 1-hepatocarcinogenesis.
M. Chelcheleh, A. Allameh / Mech. Ageing Dev. 78 (1995) 189-196
195
In addition to the enzyme-mediated activation as mentioned above, two other factors may in part be responsible for the differential AFBl-DNA residual adducts between the two age-groups. These factors include: faster rate of cell multiplication in netonatal organs which may increase the opportunities for the organ to correct the damage. In addition to this, liver is known to possess enhanced excision repair activity of several types for the adducts of various carcinogens with DNA [26]. Although much less is known about DNA repair enzymes, it seems that the repair capacity in neonatal liver is greater than that of the adults. This difference in the repair system can operate in the faster removal of the AFBl-DNA adducts from neonatal tissue (Fig. 1). Binding of the carcinogen to the cellular proteins which is indicative of aflatoxin dihydrodiol binding to lysine residues, was also affected by the lower rate of AFBl activation in newborn tissue. The ratio of DNA to protein interaction in both age groups shows that, irrespective of age, AFBl always attacks DNA at a much higher rate (Fig. 2). Active participation of lung as an extrahepatic organ in the metabolic activation of AFBl is ruled out since the level of AFBlDNA binding in lungs is significantly much lower than that in liver (Table 2, Fig. 2). The aforementioned data, mainly support the susceptibility of neonatal rat to the biological effects of AFBl. However, overall results indicate that there is a regulated mechanism by which immature animals protect their cellular macromolecules against the adverse effect of the carcinogen. On the one hand, bioactivation of the carcinogen in newborn liver slowly increases the deposition of the carcinogen in tissues and on the other hand, it gives enough time for the GSH system to inactivate the reactive metabolites formed. The slow rate of epoxidation in neonatal tissue is associated with the lower rate of AFBl-DNA residual bindings. One important factor which facilitates detoxification of AFBl in neonatal rat is that considerably higher amounts of aflatoxins are eliminated through kidneys at this age. Further studies are needed to assign the role of these factors in the detoxification of chemical carcinogens by neonatal animals. Acknowledgements This work was supported by a grant from Tarbiat Modaress University, Tehran, Iran. We express our appreciation to Dr Manju Saxena and Dr F.S. Karami Tehrani for their critical review of the manuscript. References 111W.F. Busby, Jr. and G.N. Wogan, Aflatoxins. In C.E. Searle (Eds.), Chemical Carcinogens, ACS Monograph
182, American Chemical Society, Washington, DC, 1984, pp. 945-l 136.
121D.H. Swenson, J. Lin, E.C. Miller and J.A. Miller, Aflatoxin Bl-2,3-oxide as a probable intermedia.te in the covalent binding of aflatoxin Bl and B2 to rat liver DNA and ribosomal RNA in vivo. Cancer Rex, 37 (1977) 172-181. 1313.M. Essigman, R.G. Croy, R.A. Bennet and G.N. Wogan, Metabolic activation of aflatoxin Bl: patterns of DNA adduct formation, removal, and excretion in relation to carcinogenesis. Drug Metab. Rev., 13 (1982) 581-602.
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[4] SM.
Lamplugh, R.G. Hendrickse, F. Apeagyei and D.D. Mwanmut, Aflatoxins in breast milk, neonatal cord blood and serum of pregnant woman. Br. Med. J., 296 (1988) 968. [S] A. Allameh, M. Saxena and H.G. Raj, Interaction of aflatoxin Bl metabolites with cellular macromolecules in neonatal rats receiving carcinogen through mother’s milk. Carcinogenesis, lO( 1989) 2131-2134. [6] M. Behrozikha, M. Saidee and A. Allameh, Comparison of aflatoxin Bl-DNA binding and glutathione conjugation by liver preparations from rats of different ages. Cancer Lett., 66 (1989) 69-76. [7] J. Sedlak and R.H. Lindsay, Estimation of total protein with bound and non-protein sullhydryl groups in tissue Ellman’s reagent. Anal. Biochem., 25 (1968) 192-205. [8] O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent. J. Biol. Chem.. 193 (1951) 265-275. [9] K. Burton, A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of DNA. Biochem. J., 62 (1956) 315-323. [lo] J.H. Parish, Principles and Practice of Experiments With Nucleic Acids, Longman, Harlow, 1972. 173 PP. [l I] A. Allameh, M. Chelcheleh, E. Hajizadeh and M.J. Rassaee, Comparison of the pattern of distribution of aflatoxin Bl in adult and newborn rat. Med. J. Iran (1995) in press. [12] E.C. Miller, Some current perspectives on chemical carcinogenesis in humans and experimental animals. Presidential address. Cancer Rex, 38 (1978) 1479- 1496. [13] W.K. Lutz, In vivo covalent binding of organic chemicals to DNA as a quantitative indicator in the process of chemical carcinogenesis. Mutat. Res., 65 (1979) 2899356. [I41 W.K. Lutz, Quantitative evaluation of DNA binding. Data for risk estimation and for classification of direct and indirect carcinogen. J. Cancer Res. C/in. Oncol., 112 (1986) 85-91. [I51 R.C. Garner and C.M. Wright, Binding of [‘4C]aflatoxin Bl to cellular macromolecules in rat and hamster. Chem. Biol. Interact., II (1975) 123-137. [16] SC. Booth, Bosenberg, R.C. Garner, P.J. Hertzog and K.C. Narpoth, The activation of aflatoxin Bl in liver slices and in bacterial mutagenicity assays using liver from dilferent species including man. Carcinogenesis, 2 (1981) 1063-1068. [I71 R.H. Dashwood, D.N. Arbogast, A.T. Fong, C. Pereira, J.D. Hendricks and G.S. Bailey, Quantitative
interrelationships between aflatoxin Bl carcinogen dose, target organ, DNA adduction and final tumor response. Carcinogenesis, IO (I 989) I75 - 18I. [18] P.D. Lotlikar, EC. Jhee, S.M. Insetta and M.S. Clearheld, Modulation of microsome-mediated aflatoxin Bl binding to exogenous and endogenous DNA by cytosolic glutathione S-transferases in rat and hamster livers. Carcinogenesis, 5 (1984) 269-276. [19] H.S. Ramdsell and D.L. Eaton, Species susceptibility of aflatoxin Bl carcinogenesis: comparative kinetics of microsomal biotransformation. Cancer Res., 50 (1990) 615-620. [20] H.G. Raj, M.S. Clearfield and P.D. Lotlikar. Comparative kinetic studies on aflatoxin Bl-DNA binding and aflatoxin Bl-glutathione conjugation with rat and hamster in vivo. Carcinogenesis, 5 (1984) 879-884. [2l] P.D. Lotlikar, S.M. Insetta, P.R. Lyons and E.C. Jhee, Inhibition of microsome-mediated binding of aflatoxin Bl to DNA by glutathione S-transferase. Cancer Lett., 9 (1980) 143-149. [22] G.H. Degen and H.G. Neumann, Differences in aflatoxin-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin Bl-epoxide. Carcinogenesis, 2 (1981) 299-306. [23] K. O’Brien, E. Moss, D. Judah and G. Neal, Metabolic basis of the species difference to aflatoxin Bl-induced hepatotoxicity. Biochem. Biophys. Res. Commun., 114 (1983) 813-821. [24] T.W. Kensler, P.A. Egner, N.E. Davidson, B.D. Roebuck, A. Pikul and J.D. Groopman, Modulation
of aflatoxin metabolism, aIlatoxin-N7-guanine formation and hepatic tumorogenesis in rats fed ethoxyquin: role of induction of glutathione S-transferase. Cancer Res., 5 (1986) 3924-3931. [25] H.G. Mandel, M.M. Manson, D.J. Judah, J.L. Simpson, J.S. Green, L.M. Forrester, CR. Wolf and G.E. Neal, Metabolic basis for the protective effect of antioxidant ethoxyquin on aflatoxin Bl hepatocarcinogenesis in the rat. Cancer Res., 47 (1987) 5218-5223. [26] H.E. Krokan, L.C. Olsen, R. Aasland, G. Volden, G. Eggset, B. Myrnes, B. Johnsen, A. Hangen and D.E. Hellande. In B.M. Sutherland and A.D. Woodhead (Eds.), DNA Repair in Human Tissues, Plenum, New York, 1990, pp. 175-190.
ELSEVIER
anddWel!lpment
78 (1995) 189-196
In vivo biotransformation of aflatoxin Bl and its interaction with cellular macromolecules in neonatal rats Mohamadreza
Chelcheleh, Abdolamir Allameh*
Department of Biochemistry, School of Medical Sciences, Tarbiat Modaress University, P.O. Box 14155-4838, Tehran, Iran
Received 8 June 1994; revision received 12 October 1994; accepted 11 November 1994
Abstract In this study, the ability of neonatal rat liver to metabolise [3H]aflatoxin Bl (AFBl) was compared to that of the adult animal. In order to make this comparison, neonatal and young adult rats were killed 2, 6, 12 and 24 h after injection with a single i.p. dose of AFBl. The rate of AFBl adduct formation to nuclear DNA and protein was measured in hepatic and pulmonary tissues. The results demonstrated that AFBl was epoxidized more rapidly by the adult’:; liver and lungs 2 h after the toxin administration, compared with those of the neonatal’s (adult 30 pmol and neonatal 12 pmol AFBl bound/mg DNA). However, these differences were more pronounced in hepatic than in pulmonary tissues. The same differences between AFBl-DNA adducts were also observed at different time points. These changes are certainly related to the level of hepatic cytochrome P-450. The delayed cytochrome P-450-dependent AFBl activation in neonatal’s liver provides time enough for de-epoxidation of slowly generated epoxide. The rate of AFBl-epoxide formation at this age was consistent with the activity of phase II metabolism of AFBl (glutathione conjugation). In addition, the hydrolysis of AFBl-DNA adducts at a relatively higher rate by neonatal’s liver may also contribute to the quick removal of the adducts. In spite of the aforementioned evidence which shows the capability of neonatal liver to handle AFBl, the fate of large amounts of free (non-metabolised) AFBl deposited in neonatal’s liver is not well understood.
Keywor&: Aflatoxin Bl; Neonatal;
Biotransformation;
DNA; Protein
* Corresponding author. 0047-t~374/95/$09.50
0 1995 Elsevier Science Ireland Ltd. All rights reserved
SSDZ 0047-6374(94)01536-U
190
M. Chelcheleh, A. Allameh / Mech. Ageing Dee. 78 (1995) 189-196
1. Introduction Aflatoxin Bl (AFBl) is a mycotoxin produced as a secondary metabolite by the toxigenic strains of Aspergillusflavus and A. parasiticus. It is a natural contaminant of the food chain and is a potent hepatocarcinogen in several animal species -[I]. AFBl exerts its biological effects after activation by cytochrome P-450-dependent monooxygenase to its reactive form, AFBl 2,3-epoxide, leading to covalent binding to the cellular macromolecules, particularly DNA [2,3]. In recent years, it has been observed [4] that, like adults, infant humans are exposed to AFBl and congeners. Furthermore, detection of aflatoxins in the cord blood and newborn serum indicated the transfer of these toxins to the neonatal animals. We have already studied the distribution of transmammary AFBl hydroxylmetabolites in neonatal tissues consuming milk from mothers treated with AFBl. In addition, interaction between these metabolites and cellular macromolecules such as DNA, RNA and proteins have been quantitated [5]. This was an approach for the evaluation of the ability of a neonatal rat to handle a chemical carcinogen. However, in this study the mother’s liver is believed to play the central role in the biotransformation of the carcinogen. Therefore, in this study it was difficult to rationalize the part played by the newborn liver in the metabolism of AFBI. A more recent report from our laboratory [6] clearly showed that xenobiotic metabolizing factors are underdeveloped in neonatal’s liver. Microsomal cytochrome P-450-mediated epoxidation of AFBI as measured by its binding to calf thymus DNA in vitro and inhibition of AFBl-DNA binding by cytosolic glutathione (GSH) S-transferase in the reconstitution experiments also showed the low efficiency of neonatal’s liver in metabolising the toxin. The present report is an in vivo comparative study of the AFBl biotransformation in neonatal and adult animals. AFBl adduct formation and removal to the cellular DNA and protein was studied in neonatal rats at different intervals of time to examine the capacity of neonatal tissue in hydrolysing the already formed adducts. The role of the detoxification pathway in controlling the AFBl-DNA binding in neonatal’s liver was evaluated by depleting the GSH level in this tissue prior to carcinogen administration. In addition to this, the possible role of lungs as an extrahepatic organ in sharing the metabolism of AFBI was also investigated.
2. Materials and methods 2.1. Chemicals
[3H]AFB1 was purchased from American Radiolabled Chemical Co., St. Louis, MO. Non-radioactive AFBl, diethyl maleate (DEM), reduced glutathione (GSH), bovine serum albumin, calf thymus DNA, RNA, RNAase, PPO, POPOP, DMSO and DTNB [5,5-di-thio bis-(2-nitrobenzoic acid)] were the products of Sigma Chemical Co., St. Louis, MO. All other reagents and solvents were obtained from E. Merck, Germany.
M. Chelcheleh, A. Allameh / Mech. Ageing Dev. 78 (1995) 189-196
191
2.2. Animals and treatment In this study, albino rats of Wistar strain were selected. Breeding was carried out at the animal house of the School of Medical Sciences at this university. Wherever indicated in the text, adult rats means young adult male rats, 3-5 months old. Neonatal rats were selected on the basis of their birth date and were 17 f 5 days old. All the newborn rats were determined to be male. In each experiment, four young adult rats along with lo-12 neonatal rats were selected for AFBl-treatment. Each rat was administered intraperitoneally (i.p.) with a single dose of [3H]AFB1 (specific activity 18 mCi/mmol) and sacrificed at different time intervals. Tissues from 3-4 newborns were pooled and processed for further studies. In order to compare the pattern of distribution of aflatoxin metabolites in different tissues of adult and neonatal animals, liver, kidneys, lung, intestine and stomach were removed, homogenized and radioactivity was measured. Similarly radioactivity was measured in serum samples obtained from animals prior to sacrifice [ll]. Aliquots of the lung and liver homogenate were taken for the extraction of DNA, RNA and proteins. Extraction and quantitation of AFBI adduct formation to these macromolecules was performed as described earlier [S]. 2.3. Hepatic glutathione depletion by DEM In a set of experiments, the level of cellular hepatic GSH was depleted by i.p. administration of a single dose of DEM (0.6 ml/kg body wt.) 2 h prior to AFBl administration as described above. The level of hepatic GSH was measured using Ellman’s reagent according to Sedlak and Lindsay [7]. 2.4. Other assays Protein levels were measured by Biuret reaction using bovine serum albumin (BSA) as standard [8]. DNA was estimated calorimetrically using diphenylamine reagent based on the Burton method [9]. The amount of RNA associated with the isolated DNA was < 10% as measured by Orcinol reagent according to Disch’s procedure [lo]. 2.5. Statistical analysis Statistical comparison between neonatal and adult animals was performed by non-parametric Wilcoxon and Kruskal-Wallis tests using the SPSS computer program. P-values lower than 0.05 (P < 0.05) were considered significant. 3. Results Ad.ministration of a single i.p. dose of [3H]AFB1 (8 ,uCi [3H]AFB1 containing 40 pg AFBljlOO g body wt.) to neonatal and young adult rats resulted in a differential pattern of distribution of AFBl-metabolites in various tissues [I 11. Hepatic and pulmonary tissues obtained from t3H]AFBl-treated animals were further processed for measurement of AFBl-DNA binding in both the age-groups (Table 1). The data presented in this table illustrate that AFBl adduct formation to DNA and proteins is significantly higher in adult compared to neonate’s liver.
192
M. Chelcheleh, A. Ailameh / Mech. Ageing Del). 78 (1995) 189-196
Table 1 Comparison of AFBl interaction with tissue DNA and protein in adult and neonatal rat: 2 h after AFBl treatment Tissue
Age group
AFBl-macromolecule DNA
Liver Lung
A NN A NN
119 * 34 k 3.9 * 2.9 +
binding Protein
3.4* 2.4 0.4* 0.2
4.8 * 1.1* 1.6kO.6 0.37 + 0.03* 0.27 k 0.03
A, adult; NN, neonatal; results are expressed as mean f S.E.M obtained from four samples from each group; experimental details are as described in ‘Materials and methods’ section; data are in terms of pmol [“H]AFBl bound per mg DNA. *P < 0.05 considered significant.
Low levels of AFBl adduct formation to lung macromolecules were not comparable to that of the liver in both age groups. Table 1 shows that AFBl-protein adduct was always considerably less than the adducts formed to nuclear DNA (adult 4.8 and neonatal 1.6 pmol AFBl bound/mg protein). The level of nuclear RNA isolated from these tissues was extremely low and not sufficient for measuring AFBl-RNA binding. Depletion of GSH by DEM to about 50% in neonatal liver caused an approximately 2.5-fold increase in AFBl-DNA binding. The role of neonatal hepatic glutathione in the AFBl adduct formation to DNA and protein is demonstrated in Table 2. The results of time-course adduct formation and removal of AFBl to nuclear DNA in adult and neonatal rats is compared in Fig. 1. Maximum binding in both the age groups occurred at the same time (6 h after AFBl treatment). The binding of AFBl to the adult liver DNA is not only higher, but it also found to persist longer than in the neonatal tissues (Fig. 1). It is interesting to note that the amount of residual bindings remaining after 24 h of AFBl treatment was considerably lower in the case of neonatal liver (adult 30 and neonatal 12 pmol per mg DNA). Similarly AFBl-protein interaction in adult liver was observed to be higher than that of the neonatal rats (Fig. 2). Table 2 Effect of cellular GSH-depletion on AFBl-macromolecule Treatment
Control DEM
GSH concentration umol/g liver
5.2 k 0.05* 4.2 k 0.09
binding in neonatal rat liver
AFBl-binding DNA
Protein
68.5 + 3.0 167 & 8.8*
1.5 + 0.06 3.0 f 0.09*
Data are expressed as mean k S.E.M of four samples each pooled from 3-4 livers in each group; non protein GSH concentration in liver homogenate was measured according to the Sedlak and Lindsay method [7]. *P < 0.05 considered significant.
M. Chelcheleh, A. Allameh / Mech. Ageing Dev. 78 (1995) 189-196
193
PmoVmg DNA l4CI )
t 1
I
5
10
f I
I
I
16
20
26
hours -
Adult
-+
Newborn
Fig. 1. Kinetics of the persistence of AFBl-DNA adduct in newborn and adult rat liver. Animals receive,d a single i.p. dose of [3H]AFBl and were sacrificed at different time intervals after treatment. AFBl-DNA binding is expressed as pmol [‘HIAFBI bound to milligrams of DNA. Values represent the mean + S.E.M. of three tissue samples.
4. Dixussion
The molecular niechanism(s) of hepatic preneoplastic formation due to AFBl treatment is well understood. Important determinants in the susceptibility of AFBl hepatocarcinogenesis include the enzyme-mediated activation, detoxification and PmoVmg Protein 61
Oi 0
,
I
10
5
I
I
J
16
20
25
hours -
Adult
-J-
Newborn
Fig. 2. Kinetics of [3H]AFBl-protein binding in adult and neonatal rat liver. Experimental details including animal treatments are as described in Fig. 1 legend. Values represent the mean + S.E.M. of three samples.
194
M. Chelcheleh,
A. Allameh / Mech. Ageing Dev. 78 (1995) 189-196
AFBl-DNA adduct formation and removal (repair) [12- 141. There is a good correlation between the level of covalent AFBl-DNA binding in liver slices with the susceptibility of AFBl-induced carcinogenesis in vivo in various animal species [15-171. Accordingly, laboratory animals have been classified as susceptible (rat) and resistant (hamster and mice) species based on their relative extent of biotransformation of AFB 1 [ 18-201. The susceptibility varies greatly between the different species, however within species susceptibility differs with regard to sex, age and nutritional status. The extent of the influence of age on the AFBl-induced hepatocarcinogenesis is not well understood. The presence of some important determinants of susceptibility in neonatal rats such as the difference in the rate of AFBl-DNA adducts in neonatal and adult organs in vivo indicates that the metabolic activation of AFBl is less efficient in neonatal liver. The in vitro microsome-mediated binding experiments clearly showed that, in neonatal liver as in adults, cytochrome P-450 is mainly responsible for the metabolic activation of AFBl. It has been well established that epoxidation alone cannot account for differences in AFBl-DNA binding, since the cytosolic GSH S-transferase plays a major role in the modulation of hepatic AFBl-DNA adduct formation [18,21251. Therefore, due to the reduced level of GSH and GSH S-transferase activity in neonatal liver, the rate of AFBl-GSH formation is also low at this age. Correlation between the depletion of the cellular GSH content in neonatal liver and the increased level of AFBl adduct formation to the nuclear DNA and proteins (Table 2), further substantiates the role of the GSH conjugation system in scavenging the AFBl-epoxide formed in vivo. Because of this close association between the two phases of the metabolism, it is likely that, though often below normal levels, the activity of the GSH system appears sufficient for the small amounts of the epoxide formed in neonatal liver during the phase I metabolism. An important factor which contributes to the susceptibility of neonatal rat is the deposition of large amounts of free AFBl (non-metabolised) in liver at this age. It means that the carcinogen is partially metabolised in immature liver. Hence the complete metabolism of AFBl by neonatal tissue relies on the age-dependent development of the key factors involved in the biotransformation of AFBl. These results raise the question whether alternative mechanisms are present by which the damage to cellular macromolecules caused by AFBl in neonatal rat could be prevented or delayed. The results of time-course AFBl adduct formation and removal to the liver nuclear DNA presented in Fig. 1 reveal that the main reason for the reduced AFBl-DNA interaction is the cytochrome P-450 content in the neonatal tissue. The difference in the catalytic activity of the enzyme in neonatal and adult animals in the presence of AFBl remains almost constant at different time intervals. The adducts measured 24 h after AFBl administration which are considered as residual bindings are believed to be responsible for AFB 1-hepatocarcinogenesis.
M. Chelcheleh, A. Allameh / Mech. Ageing Dev. 78 (1995) 189-196
195
In addition to the enzyme-mediated activation as mentioned above, two other factors may in part be responsible for the differential AFBl-DNA residual adducts between the two age-groups. These factors include: faster rate of cell multiplication in netonatal organs which may increase the opportunities for the organ to correct the damage. In addition to this, liver is known to possess enhanced excision repair activity of several types for the adducts of various carcinogens with DNA [26]. Although much less is known about DNA repair enzymes, it seems that the repair capacity in neonatal liver is greater than that of the adults. This difference in the repair system can operate in the faster removal of the AFBl-DNA adducts from neonatal tissue (Fig. 1). Binding of the carcinogen to the cellular proteins which is indicative of aflatoxin dihydrodiol binding to lysine residues, was also affected by the lower rate of AFBl activation in newborn tissue. The ratio of DNA to protein interaction in both age groups shows that, irrespective of age, AFBl always attacks DNA at a much higher rate (Fig. 2). Active participation of lung as an extrahepatic organ in the metabolic activation of AFBl is ruled out since the level of AFBlDNA binding in lungs is significantly much lower than that in liver (Table 2, Fig. 2). The aforementioned data, mainly support the susceptibility of neonatal rat to the biological effects of AFBl. However, overall results indicate that there is a regulated mechanism by which immature animals protect their cellular macromolecules against the adverse effect of the carcinogen. On the one hand, bioactivation of the carcinogen in newborn liver slowly increases the deposition of the carcinogen in tissues and on the other hand, it gives enough time for the GSH system to inactivate the reactive metabolites formed. The slow rate of epoxidation in neonatal tissue is associated with the lower rate of AFBl-DNA residual bindings. One important factor which facilitates detoxification of AFBl in neonatal rat is that considerably higher amounts of aflatoxins are eliminated through kidneys at this age. Further studies are needed to assign the role of these factors in the detoxification of chemical carcinogens by neonatal animals. Acknowledgements This work was supported by a grant from Tarbiat Modaress University, Tehran, Iran. We express our appreciation to Dr Manju Saxena and Dr F.S. Karami Tehrani for their critical review of the manuscript. References 111W.F. Busby, Jr. and G.N. Wogan, Aflatoxins. In C.E. Searle (Eds.), Chemical Carcinogens, ACS Monograph
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