Effects of vitamin E on liver DNA

Cancer Letters, 25 (1984) 163--170

163

Elsevier Scientific Publishers Ireland Ltd.

EFFECTS OF VITAMIN E ON LIVER DNA

I'ATRIZIA R U S S O a, M A U R O P A L A a, SILVIO P A R O D I c, C A R L A N I C O L E T T A F E R R A R I b and G I O R G I O VIDALI b

G H I A R A d,

aChemical Carcinogenesis Laboratory, bMolecular Biology Laboratory, Istituto Nazionale per la Ricerca sul Cancro, cChemical Carcinogenesis Laboratory, istituto di Oncologia, Universitd de Genora and dScientific Laboratory, XIII Unitd Sanitaria Locale, Genova (Italy) (Received 26 July 198.1) (Revised version received 25 October 1984) (Accepted 25 October 1984)

SUMMARY

Vitamin E, both in the form of dl-a-tocopherol and dl-a-tocopheryl acetate, was capahle of inducing an increased ,alkaline elution rate of liver DNA from rats treated i.p. with the vitamin. This activity was clearly both dose- and time-dependent. A statistically significant effect was observed at dosages (1.25--5.00 mg/kg) that are in the range of biological activity of tile vitamin in the rat (reabsorption-gestation bioas.~ay). Moreover, the effect was observed at dosages that are clearly not toxic. An increased alkaline elution rate of DNA is usually interpreted as suggestive of DNA damage, ilowever recent observations seem to indicate that functional modifications of chromatin packaging can also affect the elution rate of DNA.

INTROI) UCTION

Absorption of dl-a-tocoptlerol in mammals is thought to take place mainly via the lymphatic system where it is transported as part of a lipoprotein complex [3]. The site for temporary storage is the liver [4], whereas adipose tissue and skeletal muscle become qualitatively more important as time elapses following parenteral administration of the vitamin. Studies on the distribution in subcellular particles have suggested that in liver and spleen cells, dl-a-tocopherol is mainly associated with structural components of tile cells such as mitochondria, microsomes and nuclei [8]. Other investigations suggest that the incidence of cancer, following administration o f 3-methyl-4'-dimethylaminobenzene [ 18] and methylcholanthrene [5] can be significantly lowered by adding vitamin E to the diet of the treated animals. 0304-38351841$03.00 9 198.1 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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We have recently begun studies on the effects of vitamin E on the metabolism of some carcinogens, and as a first step we evaluated posssible hepatic DNA damage induced by d/-a-tocopherol itself. llere we report the effect of vitamin E on rat liver DNA using the alkaline elution technique, a method which has been extensively used to detect alkaline DNA fragmentation related to the presence of certain types of DNA adducts [11,14 ]. MATERIALS AND METItODS

The alkaline elution technique was performed as previously reported [11]. Pharmaceutical grade d/-a-tocopherol acetate was purchased from Merck, 99% pure d/-a-tocopherol was obtained from Sigma and Hoffman LaRoche. All the above products were analyzed by thin-layer chromatography on GF-254 silica gel plates (Merck) using ethyl ether/cyclohexane ( 2 0 : 1 0 0 ) as eluent and then detected under UV light. In all cases a small fluorescent spot was barely detectable close to the running front of the eluent in addition to the major spot of tile products running in the middle of the plate. S p r a g u e - D a w l e y rats ( 1 5 0 - 2 5 0 g) were used in all the experiments. The animals were injected with the indicated amounts of vitamin diluted in 0.5 ml of olive oil. The animals were killed at the indicated times after treatment, the livers were excised and a nuclear preparation was obtained as described previously [ 111. Viability of liver cells was determined according to Page and Garvey [9]. Glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and ~-glutamyl transpeptidase (~,-GTI') were determined in the sera by standard techniques [1,17]. Livers from control and treated animals were histologically examined with hematQxylin-eosin staining. RESULTS AND DISCUSSION

In our experiments statistically significant effects could be detected with vitamin E dosage of 1.25--5.00 mg/kg, which are biologically relevant. In a typical biological assay for the activity of vitamin E (reabsorption-gestation bioassay), effects are usually observed using dosages between 1 and 2 mg/kg 17]. In dose response experiments vitamin E (d/-~-tocopherol, Sigma) induced an increased elution rate of DNA in alkali when administered to rats ,t h before termination (Fig. 1 and 'Fable 1). Increased rates of elution were dose-dependent up to a dosage of 2.5 mg/kg. A saturation of the effect or even a small decrease at dosages higher than 2.5 mg/kg was observed. A typical elution profile for these experiments is presented in Fig. 2, where the effects of vitamin E obtained from 2 different sources are illustrated. These data suggest that the phenomenon observed is probably not due to contaminants that may be present in a specific vitamin E preparation.

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dl.a-Tocopheryl acetate (Merck) was also tested under identical conditions (Fig. 2). Its effect, corrected for molecular weight was approximatively 80% of that observed for the free alcohol. The 4-h time used in the dose dependence experiments, was based on a time response experiment illustrated in Fig. 3 and Table 1. The maximum effect observed was between 4 h and 24 h and practically no effect was observed after 48 h. The fact that there was no activity after 10 min eliminates the possibility that direct contamination from ttle injection site to the liver may have occurred. Gallo:l'orres and Miller [4] reported vitamin E uptake in rat liver after i.v. injection and their pattern is similar to that reported in our time-dependence experiments via i.p. administration. The dosage employed by them (4 mg/kg of d/-a-tocopheryl acetate) was comparable to the dosage used in our studies. The maximum effect on alkaline elution was observed at times when hepatic levels of vitamin E were the highest. At 48 h, when little vitamin E remained in the liver the DNA elution profiles were similar to those in controls, suggesting that the biological effect observed is reversible within a short time [4 ]. DNA fragmentation can be the result of specific DNA damage or cell necrosis [19]. To distinguish between 2 possibilities we have determined cell viability, according to 3 different parameters. We have calculated that 89% of the liver cells remain viable after 24 h as tested by trypan blue exclusion technique in treated animals, which is quit(: close {94%} to that

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168

obtained from control animals (duplicate experiments). These results are in agreement with serological data (Table 2) whicil show that vitamin E does not m o d i f y the activity of the enzymes studied. Ilistological examination further confirm that no cell necrosis had occurred following vitamin E treatment. A mild level of cloudy swelling was observed only at 24 h and at the highest dosage tested (5 mg/kg). We have noted the maximum effect on alkaline DNA fragmentation at 4 h when no cloudy swelling was present. In conclusion no relationship could be det ect ed between DNA fragmentation and c l o u d y swelling bot h in terms of time and dosage. With regard to the validation o f the alkaline elution technique up to 1982 the m e t h o d had been already utilized for testing at least 57 different compounds, belonging to different chemical classes [11]. Compounds well known for inducing alkaline DNA fragmentation were regularly detected. We have also d o c u m e n t e d that alkaline elution technique is capable of properly detecting the ef f ect in vivo of a positive control c o m p o u n d (dimethylnitrosamine) and X-rays [10] and during the course o f our experiments we have c o n d u c t e d identical controls with dimethylnitrosamine and ionizing radiations. Considering this technique as a test for alkaline DNA fragmentation, the present results clearly indicate that vitamin E induces DNA strand breakages, even at non-toxic concentrations. It is possible, however, that changes in the elution profile induced by the vitamin may be due to an altered chromatin c o n f o r m a t i o n , which in turn modifies the rate at which DNA is eluted from the filters. Th e latter possibility has been suggested by a previous study which c o m p a r e d the alkaline elution technique to the viscosimetric technique [10]. Moreover, S t o u t and Becker [16] had observed, in liver preneoplastic nodules, 4 m o n t h s after cessation of carcinogen treatment, levels of alkaline elution rate n o t compatible with the residual am ount s of adducts. DNA strand breaks have been observed during differentiation of primary TABLE 2 EFFEC~I'S OF VITAMIN E ON SERUM ENZYMES Compounds

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4

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"r-GTP 2.63 .108.98

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(olive oil)

32.25•

3'.27 13.75•

Treated animals received 2.5 mg/kg of dl-a-toeopherol acetate. In the positive control, animal~ were treated with CCI, (0.5 ml in 0.5 ml of olive oil). The animals were killed 24 II after treatment.

169

chick myoblasts [2] and in human peripheral lymphocytes [6] and it has been speculated that changes in DNA regulation are associated with transient DNA breaks. The alterations of DNA elution profile we have observed in rat liver nuclei, following vitamin E treatment could be the result of modified patterns of gene expression, where transient DNA breaks are part of the mechanisms leading to modification o f chromatin structure and transcription. ACKNOWLEDGEMENTS

This research was supported by grants from the Consiglio Nazionale delle Ricerche n. 81.01386.96 to S:P. and n. 81. 03081.11 to G.V. We would like to thank Dr. G. Nicolb for the histological examinations. REFERENCES 1 Committee on Enzyme Scandinavian for Clinical Chemistry and Clinical Physiology

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170 14 Petzold, L. and Swenberg, b.A. (1978) Detection of DNA damage induced m vivo following exposure of rats to carcinogens. Cancer Res., 38, 1,589-159.1. 1,5 Siegel, S. (1958) Non-parametric Statistics for the Behavioral Sciences, pp. 116--127. McGraw llill, New York. 16 Stout, D.L. and Becket, F.F. (1980) Progressive I)NA damage in hepatic nodules during 2-acetylaminofluorene carcinogenesis. Cancer Res., .I0, 1 2 6 9 - 1 2 7 3 . 17 Szasz, G. (1969) A kinetic photometric method of serum -I-glutamyltranspeptidase. Clin. Chem., 1,5,124--131. 18 Walton, Y.R. and Packer, L. (1981) Free radical damage and protection: relationship to cellular aging and cancer. In: Vitamin E -- a Comprehensive Treatise, p p . . t 9 5 - 5 1 7 . Editor: L.T. Machline. Marcel Dekker, New York. 19 Warlers, R.L. and llenle, K.Y. (1982) DNA degradation in Chinese hamster ovary cells after exposure to hyperthermia. Cancer Res., .12, ,t-127--4.132.