Zinc Pretreatment Research Articles

Renal glutathione depletion and nephrotoxicity of cadmium-metallothionein in rats

Renal glutathione (GSH) concentrations were reduced approximately 80% at 4 hr after a single injection of buthionine sulfoxime (BSO) (4 mmol/kg body wt) and remained reduced for at least 16 hr in male rats. Following BSO injection, rats were injected with a nephrotoxic dose of cadmium-metallothionein (Cd-MT) (0.3 mg Cd as Cd-MT/kg body wt) and killed 1, 4, or 12 hr later. Damage to the kidney was assessed histologically and by measurement of p-aminohippuric acid (PAH) uptake into renal cortical slices. Although the renal accumulation of Cd following Cd-MT injection was significantly lower in BSO-pretreated rats as compared to nonpretreated rats, the damage to kidney was more severe. At 4 and 12 hr, both Cd-MT-induced inhibition of PAH uptake and morphological damage were significantly increased in BSO-pretreated rats. In certain experiments, the induction of renal intracellular MT synthesis by zinc pretreatment slightly decreased the renal toxicity of Cd-MT in the BSO-treated rats. The results demonstrate that although GSH depletion decreases the renal accumulation of Cd in rats injected with Cd-MT, the nephrotoxicity of Cd-MT is increased. Preinduction of MT in the kidney can only partially overcome this increase in toxicity. Therefore both GSH and intracellular MT levels can influence the renal toxicity of injected Cd-MT.

Interactions of cadmium with rat testicular interstitial cell nuclei: Alterations induced by zinc pretreatment and cadmium binding proteins

To help elucidate the mechanism by which zinc prevents cadmium-induced interstitial cell (IC) tumour formation in the rat, the subcellular distribution of cadmium in isolated ICs was studied. In both isolated intact cells and in isolated nuclei, in vivo zinc pretreatment reduced the nuclear uptake of cadmium and resulted in a decreased accumulation of cadmium in subnuclear components, including chromatic and DNA. In intact cells, in vivo zinc pretreatment increased cadmium retention in mitochondria. microsomes and cytosol. An increase in the metal content in three major cytosolic cadmium-binding fractions was also seen, including the low-molecular-weight testicular cadmium-binding protein (TCBP) previously shown to differ from metallothionein. Uptake of cadmium in association with purified TCBP into isolated IC nuclei was about double that of cadmium bound to purified hepatic metallothionein. These data suggest that zinc may prevent IC tumour formation by reducing cadmium accumulation in nuclei and subnuclear components, including chromatin and DNA. This appears to be through a direct inhibition of cadmium uptake by zinc pretreatment at the nuclear membrane and also indirectly by an enhanced retention of cadmium in non-nuclear subcellular components. These data also indicate that the absence of metallothionein in ICs may render this cell population vulnerable to cadmium by allowing increased nuclear uptake.

The Toxicity of Parenteral Copper in the Chick: Dependence on Route of Administration

Preliminary work in our laboratory suggested that the route of parenteral copper administration influences the acute toxicity of copper (Cu). The present work examined the effects of Cu given either intraperitoneally (i.p.) or intravenously (i.v.) on mortality, feed intake, peritoneal fluid and hepatic accumulation of Cu and zinc (Zn) in chicks. We also studied the potential interaction of prior Zn loading on these parameters. Four-wk-old chicks were pretreated by injection (subcutaneous) with Zn acetate (5 mg Zn/kg) or sodium acetate 24 h prior to Cu treatments. Copper as Cu acetate was given either i.p. or i.v. at a dose of 1.84 mg Cu/kg. Mortality was markedly greater in those chicks given Cu i.p. compared to those given Cu i.v. (46.1% vs. 3.9%). Zinc pretreatment had no effect on any of the parameters associated with copper treatments. The accumulation (24 h) of copper in either total hepatic tissue or cytosol was comparable for both routes of injection. However, marked differences in Cu-induced hepatic Zn accumulation were observed. Massive peritoneal fluid (41% of total plasma volume) was observed 1 to 3 h following the administration of Cu i.p. None was observed in those given Cu i.v. Thus, this effect was associated with the route-dependent mortality observed in the present study. We suggest that our data highlight the gratuitous effects of reactive agents given intraperitoneally.

Effects of glutathione depletion and induction of metallothioneins on the cytotoxicity of an organic hydroperoxide in cultured mammalian cells

The effects of glutathione depletion and induction of metallothioneins (MTs) on the cytotoxicity of t-butyl hydroperoxide ( t-BHP) were investigated in cultured Chinese hamster V79 cells. The cytotoxicity of t-BHP was enhanced with increasing duration of the pretreatment with L-buthionine-SR-sulfoximine (BSO), a selective inhibitor of γ-glutamylcysteine synthetase, and was correlated with the decrease of cell glutathione, indicating that glutathione constitutes a cellular defence against toxicity by t-BHP. Desferrioxamine, a specific iron chelator, suppressed partly inhibition of cell growth induced by t-BHP and suppressed completely the increase of the cytotoxicity caused by glutathione depletion. Butylated hydroxytoluene, a diffusible radical scavenger, showed almost the same suppressive effect as desferrioxamine. These results suggest that the cytotoxicity of t-BHP enhanced by the depletion of glutathione is attributed to an action of iron-mediated reactive radical species. Pretreatment with zinc (10 −4 M) suppressed the cytotoxicity of t-BHP that was enhanced by depletion of glutathione and the extent of suppression was paralleled with increasing duration of zinc pretreatment that correlated with increased synthesis of metallothioneins (MTs). Maximum induction of MTs also suppressed the t-BHP-induced inhibition of cell growth at 4°C in glutathione-depleted cells. These results suggest that MTs act as a scavenger for the reactive radical species which are formed in an iron-mediated manner.

Mixed Sertoli-Leydig cell tumor and rete testis adenocarcinoma in rats treated with CdCl2.

Sertoli cell tumors are, except in dogs,' rare testicular neoplasms; two have been reported in rats.' Tumors of the rete testis are even rarer. Not reported in rats, they have been described to occur spontaneously in old JCL : ICR mice,'O in Mustornys,y or in mice treated prenatally with diethylstilbestrol.' This report describes the morphology of a mixed SertoliLeydig cell tumor and a rete testis adenocarcinoma in Wistar rats [Crl : (WI)BR] from two long-term experiments on the carcinogenic potential of cadmium in males. A single subcutaneous (s.c.) injection of cadmium can induce acute vascular lesions in the testis and ischemic necrosis of the seminiferous tubules. Following resolution of the necrotic debris, periand intratubular fibrosis and mineralizations occur that may also involve the rete testis. Leydig cells, however, survive and give rise to interstitial cell t ~ m o r s . ~ The first study on cadmium carcinogenesis, started to determine the dose-response relationships for cadmium chloride (CdCl,) injected subcutaneously at approximately 8 weeks of age, involved 315 rats and lasted for 104 weeks. The second study, designed to investigate effects of zinc pretreatment on cadmium carcinogenesis, involved 364 rats for 104 weeks. Most organs and all altered tissues were fixed in neutral buffered formalin, trimmed, and processed for light microscopic investigation of hematoxylin and eosin (HE)-stained paraffin sections. Periodic acid-Schiffs (PAS) reaction with or without diastase pretreatment and the alcian blue-periodic acid-Schiff's reaction were also used. Toluidine blue-stained semithin sections were obtained from the Sertoli-Leydig cell tumor that was reembedded in epoxy resin. The mixed Sertoli-Leydig cell tumor was found in a 112week-old rat that had been treated with a single S.C. injection of 30 pmol CdClJkg and three S.C. injections of 0.1 mmol Zn-acetate 6 hours before, at the same time, and 18 hours after the CdCI, injection. At necropsy, the testes appeared atrophied. A red-brown nodule, 1-cm in diameter, was in one testis. By light microscopy, both testes had bilateral diffuse, severe peritubular fibrosis and intratubular mineralizations. One testis had a single Leydig cell tumor and the other had two separate neoplasms: a small Leydig cell tumor and a larger mixed Sertoli-Leydig cell tumor (Fig. 1). The tumor consisted mainly of variably sized and shaped tubuli expanding beyond the tunica vaginalis via the rete testis (Fig. 1). Intermixed were areas of round cells with eosinophilic cytoplasm that appeared to be of Leydig cell origin. Larger, more mature tubules were lined by spindle-shaped Sertolilike cells (Figs. 1-4) with basally oriented nuclei and pale vacuolated cytoplasm extending towards a lumen. Vacuola-

Glutathione and metallothioneins as cellular defense against cadmium toxicity in cultured Chinese hamster cells

To evaluate the protective role of cell glutathione (GSH) against the toxicity of cadmium, the effect of GSH depletion on the metal toxicity was investigated and the role of glutathione was compared with that of zinc-induced metallothioneins (MTs). A 6-h incubation of cultured Chinese hamster V79 cells with 0.2 mM L-buthionine-SR-sulfoximine (BSO), a selective inhibitor of γ-glutamylcysteine synthetase, resulted in approx. 95% depletion of GSH in the cells. The depletion of GSH did not influence the rate of cell growth, the amount of cell protein or the chromosome structure during culture for at least 24 h. Cells depleted or not depleted of GSH were challenged with (1–5) · 10 −5 M CdCl 2 for 2 h and subsequent cell growth was evaluated. The cytotoxicity of cadmium was enhanced with increasing duration of BSO pretreatment and was correlated with the decrease of cell GSH, indicating that GSH constitutes a cellular defense against toxicity by cadmium. Inducibility of MTs by zinc was investigated in cultured V79 cells. Incubation of the cells with 1 · 10 −4 M zinc acetate did not result in accumulation of MTs over the control values for up to 2 h. Thereafter, however, the synthesis of MTs increased with increasing duration of zinc treatment and an approx. 9-fold increase in the amount of MTs was observed 10 h after addition of zinc. Depletion of cell GSH by BSO did not much influence the increased accumulation of MTs by zinc. In contrast, zinc at the same concentration did not influence the level of cell glutathione up to 12 h. The cytotoxicity of cadmium was markedly reduced in the cells pretreated with zinc and the protective effect of zinc was dependent upon duration of pretreatment, being parallel with the increased accumulation of MTs. Protection of cells from cadmium toxicity by zinc pretreatment was as or a little more effective in the cells depleted of GSH as in those not depleted. Thus, glutathione appears to be an intrinsic protector against cadmium toxicity, while MTs serve as an induced cellular defense that is mobilized against heavy metal stress, but takes more than 2 h to accumulate in significant amounts. Accordingly, it is suggested that GSH and MTs have cooperative protective roles against cadmium toxicity, as an initial defense for the former and a second-stage defense for the latter.

Zinc-induced reduction in melphalan cytotoxicity: heterogeneity of response of cloned human tumor cells.

Previous studies with cultured human normal fibroblasts indicated that pretreatment of the cells with zinc for 12 h prior to exposure to the alkylating agent melphalan increased survival by seven- to ninefold over survival values obtained in cultures treated with drug only. Comparable pretreatment of cells derived from a variety of human tumors resulted in an increase in survival of 1.7-fold or less. To determine whether the limited responsiveness to zinc represented a general property of tumor cells (which would be characterized by a lack of highly zinc-responsive subpopulations contained within the parental tumor populations), a series of clones was prepared from the A101D human melanoma line and the A549 human alveolar cell carcinoma line. Cells from each clone were then challenged with melphalan with and without zinc pretreatment. Twenty-five percent of the tumor clones exhibited increased resistance to melphalan following pretreatment with zinc (range of 2.1- to 5.2-fold increase in survival), indicating that the parental tumor lines were highly heterogenous in regard to inducibility to a state of reduced sensitivity to melphalan. There was no evidence of a relationship between zinc-induced reductions in toxicity and induced elevations in total intracellular glutathione content, indicating that the primary effect of zinc is not directed toward elevating intracellular levels of glutathione.

Protective effect of zinc against ethanol toxicity in mice.

Protection against the lethal effects of ethanol at 4.5 g/kg administered acutely was maximal when zinc was administered 60 min prior to ethanol. The timing of ethanol administration corresponded with elevated plasma levels of absorbed zinc. Protection was inversely related to the dose of zinc employed, as 0.5 mumol provided greater protection than 1.0 mumol, which provided greater protection than 2.0 mumol. Protection against ethanol lethality was greater if zinc was administered 60 min prior to each injection of ethanol. Acute zinc pretreatment did not alter the activity of liver alcohol dehydrogenase (ADH), nor did it alter the blood clearance of ethanol. Chronic zinc administration as ZnCl2, 100 micrograms/ml in the drinking water for 30 d, produced a 25% decrease in hepatic ADH activity, which was accompanied by a similar decrease in the intravascular clearance of ethanol.

Amelioration of bromobenzene hepatotoxicity in the male rat by zinc

Experiments were conducted to examine the role of zinc in the prevention of bromobenzene hepatoxicity in male rats. Bromobenzene (BB) (7.5 mmol/kg, ip) produced a marked hepatotoxicity as evidenced by increases in plasma alanine aminotransferase (ALT) and aspartate aminotransferse (AST) activities and a marked depression in hepatic glutathione (GSH) content 24 hr after administration. The administration of zinc (92 μmol Zn/kg, ip, at 48 and 24 hr prior to the bromobenzene) ameliorated the bromobenzene elevations in plasma AST (25%) and plasma ALT (50%) but did not alter the decreases in hepatic GSH. Following administration of [ 14C]BB, the radioactive label was distributed primarily in the cytosolic and lipid fractions derived from liver homogenates. Furthermore, the subcellular distribution of [ 14C]BB was not altered by zinc pretreatment. The extent of covalent binding of [ 14C]BB metabolites to hepatic tissue was significantly depressed in zinc-treated rats. Zinc induced the hepatic levels of metallothionein but [ 14C]BB did not bind to this sulfhydryl rich protein. Further experiments showed that zinc treatment depressed cytochrome P-450 content, the activity of NADPH cytochrome c reductase, and the metabolism of aniline, but not that of ethylmorphine. These studies suggest that the hepatoprotective effect of zinc against bromobenzene toxicity does not involve altered binding of the reactive toxic metabolite to glutathione or metallothionein, but it may be mediated by the inhibitory effect of zinc on the microsomal cytochrome P-450-dependent drug metabolizing system.

Protective effects of zinc acetate toward the toxicity of nickelous acetate in rats

Zinc pretreatment is known to ameliorate the acute and chronic effects of the toxic heavy metal, cadmium. However, the ability of zinc to decrease the toxicity of other metals has not been widely investigated. Therefore, this study was designed to determine the effects of zinc pretreatment on the acute toxicity of nickel. Male Fischer rats received either nickel alone (i.p.), zinc alone (s.c.), zinc plus nickel, or saline (i.p. and s.c.; controls). In the lethality studies, the dose of nickel was 115 μmol nickel/kg (as nickel acetate) while for all other studies the dose was 95 μmol nickel/kg. Zinc was given in multiple doses of 300 μmol zinc/kg (as zinc acetate) at −24, 0 and +24 h relative to nickel (total zinc dose 900 μmol/kg) for lethality studies or −24 and 0 h for studies 24 h and under in duration (total dose 600 μmol/kg). Zinc pretreatment significantly increased the 14-day survival of nickel-related rats. Zinc did not, however, prevent the reduction in weight gain over 2 weeks seen with nickel treatment. Hispathologically, at 120 h following nickel exposure, kidneys in the group receiving nickel alone generally showed moderate nephropathy (multifocal proximal tubule degeneration with necrosis) while in the zinc plus nickel group the nephropathy was generally mild. Zinc pretreatment had no apparent effect on the pharmacokinetics of nickel over 24 h as assessed by urinary excretion, blood levels or organ distribution. Zinc pretreatment also did not alter the subcellular distribution of renal nickel 6 h after nickel exposure. Enhanced synthesis of metallothionein did not appear to play a critical role in the reduction of nickel toxicity, since renal concentrations of this metal-binding protein, although elevated compared to control, were not different in rats receiving zinc and nickel or zinc alone. Zinc pretreatment did, however, have marked effect on nickel-induced hyperglycemia, reducing both the duration and severity of elevated blood glucose levels. Results of this study show that zinc can prevent some of the toxic effects of nickel and that the mechanism of this action does not appear to involve either metallothionein or alterations in the pharmacokinetics of nickel.

Surface Pretreatment of Zinc and its Adhesion to Epoxy Resins

Abstract An epoxy resin based on the diglycidyl ether of bis-phenol A cured with piperidine and containing 7 or 15% rubber toughening agent has been bonded to zinc and the adhesion measured by calculating the critical strain energy release rate, Gc, from single edge notched (SEN) and tapered double cantilever beam (TDCB) specimens. The zinc has been pretreated to give surfaces ranging from relatively flat to “microfibrous” ones covered with dendrites of the metal. The microfibrous surfaces give highest values of Gc with both modified and unmodified resins. The fracture surfaces have been studied with SEM, XPS and EPMA and the failure mode related to the joint toughness.

Zinc concentration and survival in rats infected with Salmonella typhimurium.

Percent survival was measured in male rats injected intravenously with live Salmonella typhimurium when plasma and tissue zinc levels were manipulated. Alzet pumps implanted intraperitoneally infused zinc gluconate or sodium gluconate (controls) from the onset of infection to 72 h postinfection. Plasma and tissue zinc levels were manipulated by infusing (i) 180 micrograms of Zn per h to achieve supranormal plasma and tissue zinc concentrations, (ii) 120 micrograms of Zn per h to prevent the infection-induced fall and to maintain plasma zinc levels at noninfection levels while raising tissue levels above that of infected controls, and (iii) 30 micrograms of Zn per h to increase tissue zinc levels while allowing the infection-induced decrease in plasma zinc. Preventing the fall in plasma zinc while raising liver zinc to supranormal levels enhanced rather than reduced percent survival; raising plasma and liver zinc to supranormal levels returned survival to control levels. Loading the liver with an excess of zinc without changing plasma zinc (30 micrograms of Zn per h) did not increase percent survival in the infected host. Pretreatment or administration of zinc at the time of infection led to increased percent survival compared with administration of zinc 4 h after the onset of infection.

Zinc-induced tolerance to cadmium hepatotoxicity

Pretreatment with Zn is known to produce tolerance to several toxic effects of Cd. This study was designed to determine if zinc pretreatment decreased Cd-induced lethality and hepatotoxicity. Rats given 4.0 mg Cd/kg, iv, died within 10 to 20 hr while there was no mortality in rats pretreated with Zn (12 mg Zn/kg, sc, 48 and 24 hr prior to Cd challenge). Ten hr after Cd, plasma aspartate aminotransferase and sorbitol dehydrogenase activities were markedly elevated and extensive histopathologic lesions of the liver were evident in control rats while such injury was not evident in Zn-pretreated rats. To examine the mechanism of this tolerance, distribution of Cd to 14 organs and the subcellular distribution in 6 organs (liver, kidneys, intestines, heart, spleen, and testes) was determined in control and Zn-pretreated rats. Two hours after challenge (3.5 mg Cd/kg, iv, 7 μCi 109Cd/mg Cd), the distribution of Cd to the liver markedly increased after Zn pretreatment without concomitant decreases in other tissues. Zn pretreatment resulted in distribution of more Cd to hepatic cytosol and less associated with endoplasmic reticulum. Gel filtration chromatography indicated that most cytosolic Cd was bound to metallothionein. These data suggest that Zn pretreatment reduces Cd-induced hepatotoxicity which prevents the lethal effects of Cd possibly by altering the hepatic subcellular distribution of Cd.

Effect of zinc pretreatment on mercuric chloride-induced lipid peroxidation in the rat kidney

The effect of zinc on mercuric chloride-induced lipid peroxidation in the rat kidney was investigated. The rats received zinc acetate (2.0 mmol/kg, po) for 2 days before being given mercuric chloride (15 μmol/kg, sc) and were killed 6, 12, and 24 hr after the last injection. Lipid peroxidation occurred in the rat kidney 12 hr after mercury administration, and this mercury-induced lipid peroxidation was significantly reduced by zinc pretreatment. A decrease in vitamin C and E contents in the kidney was observed 12 hr after the administration of mercury, and this decrease was prevented by zinc pretreatment. In the kidney of rats pretreated with zinc, the activities of the protective enzymes, glutathione peroxidase and glucose-6-phosphate dehydrogenase, were increased after mercury injection. Non-protein sulfhydryl content (mostly glutathione) also rose markedly. The results indicate that zinc not only induces metallothionein, but also increases protective enzyme activities and glutathione content, which would tend to inhibit lipid peroxidation and suppress mercury toxicity.

Influence of zinc, D-penicillamine and oxygen on poisoning with Amanita phalloides. Zinc accelerates liver regeneration and prevents the depletion of brain noradrenaline caused by the mushroom.

Pretreatment with zinc aspartate increased the survival rate of mice poisoned with a lyophilisate from the mushroom Amanita phalloides (APL). In the livers of the zinc-treated animals, confluent necrosis was cleared earlier. Zinc also prevented the reduction of brain noradrenaline caused by the mushroom as well as the rise of brain dopamine and brain weight. D-Penicillamine also protected against the lethal effects of APL. The survival afforded by zinc pretreatment was marginally ameliorated by additional oxygenation.

The urinary excretion of alkaline phosphatase after the repeated parenteral administration of cadmium to rats given a high dietary supplement of zinc

Repeated parenteral administration of cadmium (Cd) (1.5 mg Cd 2+/kg, daily for 28 days) produced an initial transitory (< 4 days) increase in the urinary excretion of alkaline phosphatase within 48 h: a second and persistent phase of enzymuria developed around day 15. Pretreatment and simultaneous administration of zinc (Zn) as a dietary supplement (2000 ppm Zn 2+) abolished the initial phase of enzymuria but had no effect upon the second phase.

Zinc nutritional status and response to lethal level of ozone exposure in rats

A number of attempts have been made to discover the chemicals which may provide protection against the lethal effects of the air pollutant ozone. The stabilizing effect of zinc on biomembranes in general is reported. It was shown in vivo and in vitro studies that lipid peroxidation in rat liver stimulated by CC1/sub 4/ was substantially decreased by zinc pretreatment. It was also shown that pretreatment of mice with a low dose of zinc (3 uM/kg body weight by gavage for 3 days prior to NO/sub 2/ exposure) prevented the development of lung edema induced by NO/sub 2/ exposure. A possibility is raised that the nutritional status of zinc in animals may affect the response of animals to lethal levels of ozone exposure. With this concept in mind a study was carried out to explore the possibility of using zinc supplementation as a means of protection of animals from ozone toxicity.

Cathodic pretreatment of zinc and zinc-coated surfaces

Cathodic pretreatment of zinc and zinc-coated surfaces

The effect of zinc and other metals on complement hemolysis of sheep red blood cells in vitro.

SummarySeveral transition series and IIB group metals were evaluated for effects on complement hemolysis of sheep red blood cells in vitro. Zinc and copper (10-4M) strongly inhibited complement activity by 83% and 66%, respectively. However, while manganese inhibited slightly (17%), neither cobalt nor cadmium had effect in this concentration range. Further results showed that zinc pre-treatment did not inactivate individual reaction components, and that zinc did not affect the membrane-lysis step. Rather, zinc exerted its effect only when added concomitantly with complement, suggesting that it interferes with the binding and/or activation steps necessary to form the complete complement complex.

A study of zinc in human teeth

Zinc accumulates in the surface structures of teeth and occurs in low concentrations in subsurface material, thus exhibiting a distributive pattern similar to that of fluoride and lead. Concentrations of zinc in surface enamel of teeth from different areas range from 430 to 2100 p.p.m. In terms of molar concentrations the concentrations are frequently of the same order as those of fluoride. In enamel the major deposition of zinc takes place before tooth eruption. In contrast to fluoride, posteruptive deposition of zinc appears to be irregular. Zinc is readily acquired by synthetic hydroxyapatite, competing with calcium for positions on the surface of the apatite crystal. Zinc pretreatment of hydroxyapatite produces a resistance to acid dissolution similar in magnitude to that produced by equivalent molar concentrations of fluoride.