Radiation-induced peculiarities of cytochrome P-450 IIE 1 and oncogenes mRNA accumulation in different rat tissues

The connection between the accumulation of c-fos mRNA and Р-450ІІЕІ m R N A (B2mRNA x , CYP2E1 mRNA) during the prereplicative period of rat liver cells division has been shown in previous work [1 ] . Now we are interested in relat ionship between on­ cogenes (c-fos and c-myc) and CYP2E1 gene exp­ ression under radiat ion influence which takes place in Chernobyl ' s exclusion zone. T h e interest is due to the key role of both C-FOS, C-MYC and P-450IIE1 proteins in cell transformation [2, 31 . In this work the attempt has been done to s tudy if any relationship takes place between the expression of these genes on RNA level after long action of low level radiat ion. Materials and Methods . Animals. Two groups of albino rats (Wistar) that have common ancestry were bred at the animal care facilities of the Inst i tute of Experimental Pathology, Oncology and Radiobiology at Kyiv and at Chernobyl ' s exclusion zone. Male rats were 4 months of age for all experiments reported here. T h e whole-body dose of radioactivity absorbed for this period by the ra ts living in Chernobyl through inhalation and feeding amounted to 1.2 ± 0.3 cGy for C s 1 3 7 . This is approximately 10 t imes higher than the dose absorbed by the control ra t s . Tissue acquisition. Different t issue samples were used from the animals grown in Kyiv (control) and Chernobyl ( i r radia ted) . T h e livers, bra ins , spleens

The connection between the accumulation of c-fos mRNA and Р-450ІІЕІ mRNA (B2mRNA x , CYP2E1 mRNA) during the prereplicative period of rat liver cells division has been shown in previous work [1].Now we are interested in relationship between on cogenes (c-fos and c-myc) and CYP2E1 gene exp ression under radiation influence which takes place in Chernobyl's exclusion zone.The interest is due to the key role of both C-FOS, C-MYC and P-450IIE1 proteins in cell transformation [2, 31.In this work the attempt has been done to study if any relationship takes place between the expression of these genes on RNA level after long action of low level radiation.
Materials and Methods.Animals.Two groups of albino rats (Wistar) that have common ancestry were bred at the animal care facilities of the Institute of Experimental Pathology, Oncology and Radiobiology at Kyiv and at Chernobyl's exclusion zone.Male rats were 4 months of age for all experiments reported here.The whole-body dose of radioactivity absorbed for this period by the rats living in Chernobyl through inhalation and feeding amounted to 1.2 ± 0.3 cGy for Cs 137 .This is approximately 10 times higher than the dose absorbed by the control rats.
RNA isolation.Total RNA from the samples of irradiated and control animals was isolated using guanidine thiocyanate by a procedure [4j and quan tified by measuring its absorbance at 260 nm; purity was assessed by determining the 260 n.m/280 nm ratio.After electrophoresis on denaturing gels, RNA integrity and loading equivalence were assessed by ethidium bromide staining.
Northern blotting and hybridization, Samples of total RNA were electroforetically size separated in 2Л M formaldehyde --1 % agarose gels \5 I The RNA was transferred to Hybond-N mem bra о is u.Amer sham», Great Britan) by a vacuum blotting method and crosslinked to the membranes using ultravioSei radiation.cDNA probes were randomly primed Ь specific activity of at least 10 8 cpm [6 ].Prehyir ridization, hybridization, and washing processes were performed as described in [7].Hybridized blots were exposed to X-ray film at -70 °С.Densitometer scans of autoradiographs were performed on an LKB I Itro scan XL Laser Densitometer.The valley-to-valley method was used to calculate baselines, and da la were normalized to the sum of signals.Ail blots were de-hybridized by incubation in H 2 0 at 90 °С for two minutes twice.They were checked for total removal of the probe by overnight exposure to X-ray film.Those blots showing total removal of the initial probe were then re-hybridized to a different cDNA clone.
Results and Discussion.The experiments repor ted here were designed to determine whether the expression of different cellular oncogenes as well as cytochrome P-450 genes is altered tn rats following the long exposure to radioactive substances absorbed by inhalation and feeding in Chernobyl's area.Pre vious work from our laboratory has shown that the dynamics of accumulation of c-fos mRNA is strongly related with that of P-450 2E1 mRNA during the prereplicative period of rat liver cells division [1].Experiments by other groups have shown modulation of c-fos mRNA following exposure of cells in culture to X-rays or other DNA-damaging agents [11][12][13], possibly leading to cell transformation and neoplasia.Fig. 1 it was shown by the microdensitometry results for c-fos oncogene.These results demonstrate the significant reduction of c-fos RNA level in almost all the tissues of Chernobyl's animals relative to controls.
Fig. 2 represents the level of c-myc RNA in different tissues of experimental and control rats.
These results demonstrate c-myc RNA accumulation in spleen and testicle cells.Expression of c-myc was somewhat reduced in liver relative to controls, while in brain it is essentially unaffected by the irradiation.This suggests that the exposure of animals to radio active substances absorbed by inhalation and feeding causes different responses on the level of RNA accumulation in different cell types, as well as selec tive modulation of specific oncogenes.It should be noted that the changes in expression observed here are relatively small.This is in keeping with experi ments from other groups demonstrating only mode rate gene modulation following exposure to DNAdamaging agents [11, [14][15][16].Since increased exp ression of some oncogenes (including c-myc) has been shown to play a role in cellular transformation [1 j, our results also suggest that one mechanism of irradiation-mediated oncogenesis may involve the mo dulation of oncogene expression in selected tissues.On the other hand, it is of great importance the decrease in activity of detoxification cell systems [17] apparently caused by the damage of detoxifying enzymes as well as their genes or mRNAs thu? enhancing the negative action of radiation on cell structures.For instance, it was recently demonstrated that low level radiation (LLR) represses enzymatic activity of superoxide dismutase [18], glutathione transferase and glutathione reductase [191 in human tissues.Our preliminary results also revealed some repression of activity of a microsomal enzyme P- 450IIE1 in rat liver under LLR influence (the data not shown).At first, the last enzyme attracted our attention during the investigation of the early period of rat liver regeneration caused by partial hepatectomy (PH).It was revealed that at the early hours of regeneration the accumulation of messenger RNA for protooncogene c-fos and gene of P-450IIE1 un dergo sharp fluctuations but in reciprocal manner [1, 20 J.The gene CYP2E1 is a member of cytochrome P-450 gene superfamily (CYP) encoding a group of hemoproteins that mostly catalyze the oxidative meta bolism of hydrophobic endogeneous compounds like steroids, fatty acids, prostaglandins and exogenous chemicals including drugs, carcinogens and environ mental pollutants [21 ].These substrates can be converted either to inert polar metabolites further eliminated in a water-soluble form or to cytotoxic or carcinogenic derivatives.Possible correlation of P-450IIE genes with malignancy was noted in some investigations 13, 22, ].Therefore It is not surprising that under the influence of radiation the expression of gene CYP2E1 essentially changes, as Fig. 3 shows.The RNA levels for this gene are very low in brain, spleen, and testicles.That is in accordance with the previously revealed liver-specific expression of CYP2E1 [23].But there is obvious discrepancy between the increasing of БІМА level and reduction of enzymatic activity of this cytochrome in liver under LLR.We suppose, it may be due to the radiationinduced mutations of this gene, as well as other damages on the way from DNA to protein.

Fig. 1 .Fig. 2 .
Fig. 1.Analysis of c-fos RNA levels in different tissues of control and irradiated animals.All micro denshomrtric data were normalized to the sum of signals: / -Kyiv; 2 -Chernobyl

Fig. 3 .
Fig. 3. Analysis of CYP2EI RNA levels in different tissues of control and irradiated animals.All microdensitomrtric data were normalized to the sum of signals: 1 -Kyiv; 2 -Chernobyl