Effects of Cadmium and Ionizing Radiation on Histones in Rat Testes

Klimová ·. , Mi‰úrová E: Effects of Cadmium and Ionizing Radiation on Histones in Rat Testes. Acta Vet Brno 2004, 73: 483-489. Changes in constituent part of the chromatin, histones, were studied in testes of rats 1-21 days after administration of cadmium (3 mg CdCl2/kg body mass i.p.) and/or whole body irradiation (3 Gy of gamma rays). It was found that administration of cadmium alone did not result in changes of histones; the ratio of H/DNA, however, decreased due to temporary increase in DNA concentration. Irradiation alone caused an initial increase in concentration and total content of histones, which was followed by gradual decrease in histone content and changes in relative proportion of individual histone fractions until the end of experiment. On day 21 after irradiation alone, also abrupt increase in H1t subfraction in H1 histone fraction was found. After combined treatment (cadmium administration 30 min before irradiation), the alterations of histones were similar as after irradiation alone but they were more expressed. This finding suggested that partial summation of the two factor effects on the testicular histones occurred after the combined treatment of rats. Cadmium, gamma irradiation, rat, testes, histones Cadmium is a highly toxic and carcinogenic environmental contaminant, which has no special biological role in animal organism (Smith et al. 1994; Koréneková et al. 2002). It is accumulated in the human body with a half-life exceeding 10 years and has been linked with a number of health problems including marked damage to testes and fertility reduction (Kelley 1999; Bench et all. 1999). Acute cadmium-induced damage to testes manifests itself by haemorrhagic inflammation, degeneration and dysfunction of the organ, morphological changes or apoptotic death of germinal cells, etc. (Xu et al. 1996; Yan et al. 1997; Zhou et al. 1999; Waalkes 2000). On the cellular level, the toxic effects of cadmium in some organs, e.g. testis, liver and brain, include alterations in permeability of plasma membranes, damage to nuclear and mitochondrial membranes, increase in chromatin condensation, ladder-like splitting of DNA and decrease in the total DNA content, respectively (Koizumi et al. 1996; Fasanyaodewumi et al. 1998; Klimová and Mi‰úrová 2001). Irreversible changes of genome may be caused by an oxidative DNA damage leading to DNA strand-breaks, DNA-protein cross-linking and inhibition of DNA repair (Oteiza et al. 1999). Many cadmium-induced changes (e.g. oxidative damage to DNA, chromatin condensation, decrease in DNA content, apoptosis) are similar to those caused by other harmful factors, especially by ionizing radiation. The most striking effects of ionizing radiation include, however, inhibition of DNA synthesis and mitosis and especially increase in the number of chromosome abnormalities (Van Buul and Goudzwaard 1990; De Rooij and Grootegoed 1998; Gobé et al. 1999). DNA is arranged in a complex with histones into a highly ordered compact and dynamic chromatin structure (Beato and Eisfeld 1997; Ren and Tong 1997; Smerdon and Concol i 1999). Histones are subjected to post-translational modifications that might ACTA VET. BRNO 2004, 73: 483-489 Address for correspondence: Prof. RNDr. Eva Mi‰úrová, CSc. ·afárik University Institute of Biological and Ecological Sciences Moyzesova 11, 041 54 Ko‰ice Slovak Republic Phone: + 421-55-6222610 E-mail: misurova@kosice.upjs.sk http://www.vfu.cz/acta-vet/actavet.htm influence the packaging of DNA in nucleosomes and higher-order structure of chromatin fibres. By this way, the histones can regulate transcriptional activity in non-specific or genespecific manner and are involved in the processes of cell proliferation, differentiation and senescence (Berger 2002; Kouzarides 2002; Oki and Kamakaka 2002). Therefore, structural alterations and chemical modulations of DNA and histones induced by genotoxic agents and mainly the degree of DNA damage repair in the nuclear chromatin are the crucial events leading to cell death or cancerogenesis. Until present, attention was paid particularly to the study of effects of ionizing radiation or other genotoxic factors on DNA structure, replication and repair of DNA damage in various tissues. Due to increasing level of some types of heavy metals and ionizing radiation in the environment, the biological effects of these agents should be considered in the context of combined exposure of an organism to multiply agents. In the present study, therefore, the alterations of histones were investigated in rat testes after administration of cadmium (3 mg CdCl2/kg body mass i.p.) and gamma irradiation (3 Gy) alone or after combined treatment. Materials and Methods The experiment was performed with 80 male rats of the SD strain (Anlab Praha, Czech Republic), 6-week-old at the beginning of experiment. They were kept under standard vivarium conditions (temperature 22-24 °C, natural light rhythm). Food and water were provided ad libitum. The animals were divided into four groups: Non-treated control rats (C) Rats treated with cadmium (Cd) Rats irradiated with gamma rays (Ir) Rats treated with cadmium before irradiation (Cd + Ir). Cadmium adminis t ra t ion Cadmium (CdCl2, Lachema Brno, Czech Republic) was administered i.p. at the dose of 3 mg⋅kg -1 body weight alone or 30 min before irradiation (groups Cd and Cd + Ir, respectively). I r radiat ion Animals were total-body irradiated with the dose of 3 Gy by gamma rays from a 60Co source (Chisostat Chirana, Czech Republic) at a dose rate of 0.150 Gy⋅min-1 (groups Ir and Cd + Ir). The experimental conditions complied with the requirements for ethical standards of welfare and treatment of animals. The treatments were arranged in such a way that the rats achieved 9 weeks of their age at the time of sacrifice. At this age, the male rats are sexually mature; in the testes, spermatogonia A, spermatogonia B, spermatocytes I and spermatocytes II divide by mitotic, resp. meiotic divisions. However, due to the long duration of the first meiotic prophase, the spermatocytes I are the most numerous cells in the organ (Keeton and Gould 1986). Isolat ion of cel l nuclei Cell nuclei were isolated according to the method of Grünicke et al. (1989). Extract ion of his tones Histones were extracted from the nuclei with 0.2 M H2SO4 for 1 hour. After precipitation and repeated washing of samples with acetone, concentration of histones was determined by the method of Lowry et al. (1951). Gel e lectrophoresis of his tones Separation and detection of individual histone fractions was carried out on polyacrylamide gels according to the method of Panyim and Chalkley (1969). After staining with amido black B, the relative proportion of the individual histone fractions was determined spectrophotometrically using a densitometer (Shimadzu CS-930, Japan). Detection of the individual fractions H2A, H2B and H3 was not always unambiguous and common values for these histone fractions (H2 + H3) are therefore given in Fig. 2. Stat is t ical analysis The experimental data were statistically evaluated by Peritz ́ F-test (Harper 1984) and they are given as mean ± SEM in the Figures and Table 1.

influence the packaging of DNA in nucleosomes and higher-order structure of chromatin fibres.By this way, the histones can regulate transcriptional activity in non-specific or genespecific manner and are involved in the processes of cell proliferation, differentiation and senescence (Berger 2002;Kouzarides 2002;Oki and Kamakaka 2002).Therefore, structural alterations and chemical modulations of DNA and histones induced by genotoxic agents and mainly the degree of DNA damage repair in the nuclear chromatin are the crucial events leading to cell death or cancerogenesis.
Until present, attention was paid particularly to the study of effects of ionizing radiation or other genotoxic factors on DNA structure, replication and repair of DNA damage in various tissues.Due to increasing level of some types of heavy metals and ionizing radiation in the environment, the biological effects of these agents should be considered in the context of combined exposure of an organism to multiply agents.In the present study, therefore, the alterations of histones were investigated in rat testes after administration of cadmium (3 mg CdCl 2 /kg body mass i.p.) and gamma irradiation (3 Gy) alone or after combined treatment.

Materials and Methods
The experiment was performed with 80 male rats of the SD strain (Anlab Praha, Czech Republic), 6-week-old at the beginning of experiment.They were kept under standard vivarium conditions (temperature 22-24 °C, natural light rhythm).Food and water were provided ad libitum.The animals were divided into four groups: Non-treated control rats (C) Rats treated with cadmium (Cd) Rats irradiated with gamma rays (Ir) Rats treated with cadmium before irradiation (Cd + Ir).

Cadmium administration
Cadmium (CdCl 2 , Lachema Brno, Czech Republic) was administered i.p. at the dose of 3 mg⋅kg -1 body weight alone or 30 min before irradiation (groups Cd and Cd + Ir, respectively).

Irradiation
Animals were total-body irradiated with the dose of 3 Gy by gamma rays from a 60 Co source (Chisostat Chirana, Czech Republic) at a dose rate of 0.150 Gy⋅min -1 (groups Ir and Cd + Ir).
The experimental conditions complied with the requirements for ethical standards of welfare and treatment of animals.The treatments were arranged in such a way that the rats achieved 9 weeks of their age at the time of sacrifice.At this age, the male rats are sexually mature; in the testes, spermatogonia A, spermatogonia B, spermatocytes I and spermatocytes II divide by mitotic, resp.meiotic divisions.However, due to the long duration of the first meiotic prophase, the spermatocytes I are the most numerous cells in the organ (Keeton and Gould 1986).

Isolation of cell nuclei
Cell nuclei were isolated according to the method of Grünicke et al. (1989).

Extraction of histones
Histones were extracted from the nuclei with 0.2 M H 2 SO 4 for 1 hour.After precipitation and repeated washing of samples with acetone, concentration of histones was determined by the method of Lowry et al. (1951).

Gel electrophoresis of histones
Separation and detection of individual histone fractions was carried out on polyacrylamide gels according to the method of Panyim and Chalkley (1969).After staining with amido black B, the relative proportion of the individual histone fractions was determined spectrophotometrically using a densitometer (Shimadzu CS-930, Japan).Detection of the individual fractions H2A, H2B and H3 was not always unambiguous and common values for these histone fractions (H2 + H3) are therefore given in Fig. 2.

Statistical analysis
The experimental data were statistically evaluated by Peritz´ F-test (Harper 1984) and they are given as mean ± SEM in the Figures and Table 1.

Quantitative changes
No quantitative changes of histones were found in the testes of rats treated with cadmium alone (Fig. 1).In rats exposed to gamma irradiation alone, a transient increase in histone concentration and total content occurred on day 1; at the following intervals, there was a gradual decrease especially in histone content.After the combined treatment a transient increase in histone concentration occurred on the 14th day; due to high variation of values, however, the increase was statistically insignificant and it was not accompanied by adequate increase in the total histone content in the organ.The changes in total histone contents were more unequivocal than changes in histone concentrations and after combined treatment they manifested themselves by decrease, which was slightly deeper than after irradiation alone and on day 21 it was accompanied also by significant decrease in histone concentration.

Histone fractions
In histones extracted from the testes of rats treated only with CdCl2, no changes were found in relative proportion of individual fractions (Fig. 2).After irradiation alone, the proportion of fraction H1 was increased from day 7 until the end of the experiment.The increase in H1 fraction was on account of H2 + H3 fractions and on day 21 also on account of decrease in H4 fraction.The combination of the two treatments induced the same changes in relative proportion of histone fractions as irradiation alone with the exception of the 14th day, when an increase in H1 fraction and loss of H4 fraction was much more conspicuous than after irradiation alone.
The proportion of H1t subfraction in the H1 fraction transiently increased on the days 1 and 14 after administration of cadmium alone (Fig. 3).After irradiation alone or in combination with cadmium, relative proportion of H1t subfraction sharply increased on day 21 after the treatments in comparison to control.Temporary decreases in H1t subfraction, which occurred at the earlier intervals, were not statistically significant.

H/DNA ratio
In all groups of experimental rats, the ratio of histones to DNA was lowered in course of experiment with exception of day 14 after treatment (Table 1).At the first two intervals of examination, the decline in H/DNA ratio resulted from temporary increase in DNA concentration, whereas decline on day 21 was mainly due to the decrease in histone concentration.

Discussion
Administration of cadmium alone (3 mg CdCl 2 /kg) did not induce changes of histones in rat testes with the exception of transient increase in H1t subtype in H1 histone fraction; an  initial drop in H/DNA ratio in this group was due to increase in DNA concentration.In the other groups of experimental rats, the changes of histones were much more profound and majority of them appeared at later periods after treatments.After irradiation with gamma rays (3 Gy) alone or in combination with cadmium, the content of histones in testes of rats gradually decreased until the end of investigation.The decrease in histone content was caused mainly by fall in H4 and H2 + H3 fractions, therefore the relative proportion of H1 fraction increased.The decrease in concentration of histones on day 21 after irradiation alone or in combination with cadmium was accompanied by abrupt increase in H1t histone subtype.It seems, therefore, that H4 fraction is the most labile fraction of histones in rat testes; the most stable fraction seems to be H1 fraction, especially its subfraction H1t.Decrease in histone content in the testes of treated rats is in agreement with findings of Datta et al. (1993) concerning the effects of ionizing radiation on various proliferating and non-proliferating tissues.Decrease in histone content without adequate decrease in DNA content can be explained by partial acetylation of some histone fractions occurring mainly in cells activated to proliferation and transcription (KoÏurková et al. 1995;R e n and Tong 1997), because acetylation leads to weakening of electrostatic bonds between DNA and histones.In our experiment, especially at later intervals, the decrease in histone content in testes was related to fall in relative proportion of H4 fraction, which is acetylated in the highest degree.Preferential decrease in H4 fraction was found after irradiation also in other rat tissues, such as intact and regenerating liver and cerebellum (KoÏurková et al. 1991;Klimová and Mi‰úrová 2003).On the other side, the alterations in total histone content and fall in relative proportion of H4 histone fraction were accompanied by increase in proportion of H1 fraction.Linker histone H1 undergoes acetylation in the lowest degree (KoÏurková et al. 1995) and after irradiation it becomes transiently dephosphorylated (G u o et al. 2000) therefore the loss of H1 histone fraction due to weakening of its binding to DNA is improbable.The increase in relative proportion of H1 fraction might be related also to changes in extrachromatin pool of histones, which almost completely consists of the H1 fraction (Zlatanova et al. 1990).
H1t represents a testis-specific subtype of H1 histone occurring only in male germinal cells at the terminal stage of differentiation (Bucci et al. 1981;Ullrich et al. 1999).Synthesis of this subtype of histones was found in zygotenic or early pachytenic spermatocytes.The increase in relative proportion of H1t subtype, which was observed mainly in the testes of irradiated rats (Ir, Cd + Ir) on the 21 st day after irradiation, can be, therefore, related to reappearance of spermatocytes at this time of regeneration of seminiferous epitelium.
In general, alterations induced by administration of cadmium (3 mg CdCl 2 /kg) alone were milder than those induced by gamma irradiation (3 Gy) alone; however, the character of alterations was similar.In majority of cases, after combined treatment (cadmium being administered 30 min before irradiation), the alterations of histones were more profound than after single treatment.These findings suggest that partial summation of the two factor effects occurs after combined treatment.