Results and discussion

Table1. Basal levels of main parameters investigated.

Parameter	Mm	Units
ALAS activity	0,0990,009	nmol ALA/min/ mg of protein
HO activity	0,0420,004	nmol bilirubin/min/ mg of protein
CytP450 content	0,1470,016	nmol/mg of protein
Heme in mitochondria in postmitochondrial	1,280,15 0,540,10	nmol/mg of protein nmol/mg of protein
TDO heme saturation	41,40,7	%
GSH content	5,40,1	mol/g of tissue
GST activity: In mitochondria In postmitochondrial	16819 89667	nmol CDNB/min/ mg of protein nmol CDNB/min/mg of protein

Both HgCl₂ and CdCl₂ were found to cause significant increase of ALAS and HO activities and decrease of CytP450 content 24 hrs after action in vivo (Fig.1, *– p<0,05 versus control values).

Both CdCl₂ and HgCl₂ were found to increase total heme content in liver first hours after action (Fig.2).

Cadmium increased heme content in PMch (Fig 2.) and microsomes (1,780,02 nmol/mg of protein versus 1,080,01 in control, p<0,05) 2 hrs after action. TDO% raises (free cytosol heme) 6 hrs after injection (60,11,1% versus 41,40,7 in control, p<0,05) and is accompanied by increase of TBA-active products content (0,730,04 nmol/mg protein versus 0,340,03 in control, p<0,05). Mercury ions caused the increase of THC in Mch and PMch (but not in microsomes, data not shown) and TDO% 2 hrs after injection (Fig.2).

The increase of THC in microsomal fraction after CdCl₂ and not after HgCl₂ action may be due to plasma protein damage and disturbances of hemopexin-mediated transport of heme to liver under HgCl₂ action. Both metals caused hemolysis and accumulation of heme-containing products in blood serum all terms after action (more than 200% to control values, control is 0,0230,002 A/mg of protein, data not shown). So one of the sources of heme in cytosol and microsomes is the heme transported from blood stream. Taking into account the possible role of membrane damage in heme redistribution and regulation of number of enzymes under heavy metals action, the pretreatment by lipophylic antioxidant and membrane stabilizer -tocopherol [13] was used 2 hrs before metal salts injection. Tocoferol pretreatment was found to prevent ALAS induction 24 hrs after both CdCl₂ and HgCl₂ action, and induction of HO 24 hrs only after HgCl₂. Tocoferol protected cytP450 from degradation 24 hrs after CdCl₂ and HgCl₂ action.

HgCl₂ was revealed to be much more potent inducer of GST than CdCl₂. Total GST activity in liver was only slightly elevated 24 hrs after Cd action (64123 versus 52616 nmol CDNB/min per mg of protein or 122% of control, p<0,05) and significantly (140085 versus 48217, or 290% of control value, p<0,05) 24 hrs after HgCl₂ action.

Significant increase of GST activity in PMch under co-administration of CdCl₂ and tocopherol may lead to enhanced utilisation of GSH and prevent elevation of GSH content under CdCl₂ action (Fig.3). Increased GST activity may provide protection of cytoplasmic components being one of the reasons of normalization of ALAS activity and cytP450 content.

The capacity of GSH, tocopherol and oxidants (H₂O₂) to influence on heme redistribution within cell was examined in vitro (Fig.4).

The inhibitive action of tocopherol on total GST activity was shown also in vivo (28339 4 hrs after tocopherol acetate action versus 66052 nmol CDNB/min per mg of protein in control, p<0,05). Hydrogen peroxide caused accumulation of heme in Mch and tendency to increase of GST activity in PMch.

Thus Hg ions action on Mch heme content may be due to decrease of GSH content and maintenance (no decrease) of ALAS activity. Increase of GSH content and inhibition of ALAS under Cd ions action may prevent mitochondrial heme accumulation (Fig.2).

The internal origin of mitochondrial heme increase was also revealed in glycerol model of rabdomyolysis in vivo. Glycerol was found to cause the increase of HO activity all terms after action and cytP450 content decrease 24 hrs after injection (Fig.5).

THC increase in PMch (Fig.6) may be partly due to heme transport from blood stream for degradation, but it is much less expressed than heme accumulation in blood. It’s known that the main consequence of glycerol action and rabdomyolysis is renal failure and metabolic disorders in kidney [2,15,16].

So we may suppose that under GSH level decrease and membrane damage conditions heme transport mechanisms are disrupted and newly synthesized heme enlarges free heme pool (according to TDO heme saturation). THC in PMch fraction remains higher control values probably due to microsomal heme because of prevention of HO induction after co-administration of glycerol and cycloheximide.