Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole.

The effect of zinc on lipid peroxidation initiated by either ferric-nitrilotriacetate, t-butyl hydroperoxide, or 3-methylindole was studied using primary monolayer cultures of rat liver parenchymal cells. The malondialdehyde content of the cells and culture medium was used to estimate the extent of lipid peroxidation. As the zinc concentration of the culture medium was increased from 1 to 48 microM, peroxidation was diminished. Cellular zinc and metallothionein levels were proportionally increased by supplemental zinc. Zinc supplementation of the medium inhibited NADPH-cytochrome c reductase activity and stimulated glutathione peroxidase activity. The uptake of iron into the hepatocytes was significantly reduced as the level of zinc was raised, suggesting that zinc antagonizes uptake of chelated iron into isolated hepatocytes and in this way blocks iron-induced peroxidation. Furthermore, induction of metallothionein synthesis by zinc may contribute to the reduction in free radicals. Spectra from electron spin resonance studies, using phenylbutylnitrone as a spin-trapping reagent, demonstrated that free radical production was inversely related to the zinc concentration of the culture medium. Spin trap data suggest that metallothionein added to lysed cells in vitro decreases free radical production. Studies using the spin trap, 3,3,5,5-tetramethylpyrroline-N-oxide indicated that cumulatively the predominant radical present in the cultures was a phenyl radical with hydroperoxide or methylindole. Collectively, our data demonstrate that zinc inhibits free radical production and lipid peroxidation in cultured hepatocytes. The mode of action of zinc could occur via free radical scavenging by zinc-induced metallothionein and/or by processes related to cytochrome P-450 and glutathione peroxidase, since these were also found to be sensitive to zinc supplementation levels of the culture medium.

Studies on the roles of apotransferrin and caeruloplasmin (EC 1.16.3.1) on iron absorption in copper-deficient rats using an isolated vascularly- and luminally-perfused intestinal preparation.

1. Studies have been made on the effects of dietary copper on the iron and Cu distribution in rats and on the metabolic activity and absorptive capacity of intestines perfused both vascularly and luminally. 2. Rats maintained for 4-5 weeks on a Cu-deficient diet (0.4 microgram Cu/kg) had significantly lower plasma, liver and intestinal Cu concentrations and significantly reduced plasma caeruloplasmin and liver cytochrome c oxidase (EC 1.9.3.1) activity compared with controls receiving a Cu-supplemented diet (5 micrograms Cu/kg). Disturbances in Fe metabolism in Cu-deficient rats were evident as shown by a mild anaemia, significantly elevated hepatic Fe concentrations and hypoferraemia. 3. Intestinal glucose uptake from both the luminal perfusion medium (LPM) and vascular perfusion medium (VPM) was unaffected by Cu deficiency despite a significant (25-30%) reduction in oxygen consumption. This was associated with a 40% decline in mucosal cytochrome c oxidase activity. 4. In studies of Fe absorption, Fe uptake from the LPM was unaffected by Cu deficiency while transfer of Fe to VPM was significantly reduced (50%) compared with control preparations. Addition of apotransferrin (1 g/l) to the VPM was without effect in preparations from control rats but significantly increased the transfer of Fe to the VPM in preparations from Cu-deficient rats without affecting Fe uptake from the LPM. 5. The addition of either human or porcine caeruloplasmin (together with apotransferrin) to the VPM, such that the resultant ferroxidase (EC 1.16.3.1) activity of the VPM supernatant fraction was four to five times that of normal rat plasma, was without effect on either Fe uptake, tissue retention or Fe transfer to the VPM by preparations from either Cu-deficient or control rats. 6. These findings offer no evidence in support of the proposed role for caeruloplasmin with its associated ferroxidase activity in Fe absorption in the rat.

Glucose uptake and iron absorption by an isolated, vascularly and luminally perfused preparation of rat small intestine.

Some characteristics of an isolated vascularly and luminally perfused rat intestinal preparation are described. Over 60 to 90 min perfusion, the vascular perfusion medium (VPM) 'arterial' pressure remained stable and between animals it varied between 22 and 47 mmHg while the VPM flow rate remained constant at 1.3 ml/min. Hypersecretion, as monitored by changes in the concentration of phenol red in the luminal perfusion medium (LPM), did not generally occur. Histological examination of the tissue post-perfusion showed that normal morphology was retained for at least 1 h. Total glucose uptake ranged between 85 and 155 mumol/(g dry wt. h) with greater uptake from the VPM than the LPM. Approximately 50% of the glucose taken up by the intestine appeared as lactate in the perfusion media, most of which was released to the VPM. The addition of phloridzin (5 x 10(-4) M) to the LPM significantly reduced the uptake of glucose from the LPM and its transfer to the VPM. Iron absorption was studied by adding FeCl2 (with 59Fe and ascorbate, 100 micrograms/ml) to the LPM to give a final concentration of 10-100 micrograms iron/ml. The appearance of iron in the VPM was investigated over a period of 1 h. At the two lowest concentrations (10 and 25 micrograms/ml) there was a lag of 20-40 min before iron appeared in the VPM. At the higher doses (50 and 100 micrograms/ml) the lag was 10 min. During the final 20 min the appearance of iron in the VPM plateaued to dose-dependent constant rates. The ratio of the specific radioactivity of 59Fe in the two perfusates (LPM:VPM) was unity when the initial iron concentration was 25 micrograms/ml or above. At the lowest dose (10 micrograms/ml) this ratio was 2.5 indicating that during transfer, iron taken up from the LPM had mixed with a small labile endogenous iron pool. Both amounts of iron retained by the intestinal tissue and transferred to the VPM increased exponentially with increasing luminal iron dose. The amount of iron retained by the tissue was always greater than that transferred to the VPM.

Regulation of liver zinc metabolism and metallothionein by cAMP, glucagon and glucocorticoids and suppression of free radicals by zinc.

Regulation of metallothionein synthesis and concomitant changes in the kinetics of zinc metabolism are influenced by dibutyryl cAMP, epinephrine, glucagon and dexamethasone in both intact rats and isolated rat liver parenchymal cells. Liver metallothionein levels were elevated many fold and were directly related to the transient hypozincemia produced following administration of these hormones or Bt2cAMP. Incubation of monolayer cultures of liver cells with these agents caused changes in both rapidly taken up 65Zn2+ and slow 65Zn2+ exchange; and increased metallothionein levels up to 7 fold. Using a 32P-labeled oligonucleotide, corresponding to a 21 base sequence of the metallothionein gene as a hybridization probe, metallothionein mRNA levels were found to be increased by each hormone and cAMP in isolated liver cells and intact rats. This indicates metallothionein gene expression is regulated by cAMP in addition to metals and glucocorticoids. Free radical damage to isolated liver cells caused by t-butylhydroperoxide or 3-methylindole increased malondialdehyde production which could be reduced by addition of Zn2+ to the culture medium. This suggests zinc uptake influenced the extent of lipid peroxidation. Spin-trapping techniques using electron spin resonance showed zinc also reduced free radical formation by the isolated hepatocytes. The concentration of metallothionein in liver cells seems to be related to cellular functions of zinc and coordinately regulated by glucagon through changes in cAMP levels and glucocorticoids.

Studies on the effects of dietary zinc dose on 65Zn absorption in vivo and on the effects of Zn status on 65Zn absorption and body loss in young rats.

Weanling male rats were maintained on diets containing 5, 10, 20, 40, 80 or 160 mg zinc/kg for 14 d. On day 15 they received 65Zn either by intraperitoneal injection or in a test meal containing 20 mg Zn/kg. After dosing, the rats were again maintained on the diets they had received previously. Whole-body 65Zn retention was measured immediately after dosing and daily for a further 9 d. From regression analysis of the semi-logarithmic plots of 65Zn retention from 0 to 192 h after 65Zn administration, the true extent of 65Zn absorption and the biological half-life (t1/2) of body 65Zn stores were calculated. At the end of the experiment, the rats were killed and the entire small intestines of some rats from each group were rapidly flushed out to remove food and faecal residues, frozen in liquid nitrogen and stored under an atmosphere of N2 at -20 degrees before separation of cytosolic Zn-binding fractions by gel filtration on Sephadex G-75. The results suggest that rats which received diets that were either deficient (5 mg Zn/kg), marginal (10 mg Zn/kg) or adequate (20-80 mg Zn/kg) in Zn achieved homeostatic regulation of body Zn by changes in both the extent of Zn absorption and excretion. However, when Zn supply was excessive, increasing from 80 to 160 mg Zn/kg, no further changes were seen in Zn absorption, and homeostatic control appeared to be effected entirely by changes in rates of body Zn loss. Gel chromatography of intestinal cytosol on Sephadex G-75 revealed that Zn was associated with two major fractions. The first (peak 1) had a molecular weight (MW) greater than 75 kdaltons and the second (peak 2), a MW of approximately 10 kdaltons and was assumed to be metallothionein. There was no obvious relation between the amount of Zn bound to peak 1 and dietary Zn content. In contrast, the amount of Zn recovered in peak 2 increased linearly with increasing dietary Zn content. Comparisons between the effect of dietary Zn content on Zn bound to peak 2 and 65Zn retention may, depending on the range of Zn intakes, indicate possible roles for intestinal metallothionein in the control of Zn absorption or excretion. A study of the effects of dietary dose of 65Zn on the extent of 65Zn absorption in rats of normal Zn status indicated a possible biphasic relation. At low doses (5-40 mg Zn/kg) 65Zn absorption appeared to exhibit a curvilinear response to increasing 65Zn dose, indicating possibly a saturable process.(ABSTRACT TRUNCATED AT 400 WORDS)