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Platelet-derived growth factor (PDGF)-, epidermal growth factor (EGF)- and insulin-like growth factor I (IGF-I)-stimulated cell proliferation in 3T3 cells was accompanied by increased abundance of labile intracellular pool of zinc (LIPZ). However, the origin and regulation of this cell proliferation-associated increase in the abundance of LIPZ are unknown. Cellular zinc homeostasis involves zinc transporters and metallothionein. The objectives of this study were to determine whether cell proliferation-associated increase in the abundance of LIPZ was a result of an increased zinc uptake and to assess the involvement of zinc transporters and metallothionein in this cell proliferation-associated increase in the abundance of LIPZ in 3T3 fibroblasts. Zinc transporters assessed included both zinc importer (Zip1) and zinc exporters (ZnT1, ZnT2 and ZnT4). Growth factors increased the abundance of LIPZ while total cellular zinc concentration remained unaffected, demonstrating that LIPZ was responsive to the increased needs for zinc during growth factor-stimulated cell proliferation. Growth factors also increased net zinc retention as indicated by higher 65zinc radioactivity and elevated mRNA levels of Zip1, ZnT1 and ZnT4. Although zinc is essential to cell proliferation, excessive cellular zinc accumulation causes cytotoxicity. Collectively, these observations suggest that increase in the abundance of LIPZ during growth factor-stimulated cell proliferation was due to increased net retention of extracellular zinc, which was apparently achieved through a coordinated up-regulation of the expression of transporters involved in both zinc influx and efflux to ensure adequate supply of zinc to sustain cell proliferation, yet to prevent potential zinc cytotoxicity in 3T3 cells.

Zinc has been shown to be accumulated in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors. Zinc is required for cell proliferation and tumorigenesis is characterized by dysregulation of cell proliferation. An accumulation of zinc in mammary tumors perhaps indicates a reliance on zinc to sustain tumor growth. Limiting zinc supply by means such as reduced zinc intake should negatively modulate mammary tumorigenesis. Our objective was to determine the effects of zinc status on MNU-induced mammary tumorigenesis in sexually mature female rats. Twenty-one-day-old Sprague-Dawley rats were assigned to low-zinc (3 mg zinc/kg diet) or adequate-zinc (12 mg zinc/kg diet) ad libitum or pair-fed control group (n = 25-33 rats/ group). On day 50 of age, all rats were intraperitoneally injected with MNU (50 mg/kg body wt) to induce mammary tumorigenesis. Rats were further maintained on their assigned diet until 14 weeks post-MNU injection. Total food intake and overall body weight gain were lower in low-zinc rats than in adequate-zinc ad libitum control rats, but were similar to adequate-zinc pair-fed control rats. Plasma zinc concentration was lower in low-zinc rats than in adequate-zinc ad libitum and pair-fed control rats, confirming moderately low-zinc status in low-zinc rats. Tumor incidence (46 versus 84 and 80%; P < 0.05) and tumor multiplicity (0.8 versus 5.0 and 2.6 tumors/rat; P < 0.05) and tumor number (28 versus 123 and 66 tumors) were reduced in low-zinc rats compared with that in adequate-zinc ad libitum and pair-fed control rats, respectively. Tumor latency in low-zinc and adequate-zinc pair-fed control rats was not significantly different, but was longer than in adequate-zinc ad libitum control rats (P < 0.05), suggesting that reduced food intake associated with low-zinc intake prolonged tumor latency. Tumor burden and size were not affected by zinc intake. Overall, our observations showed that moderately low-zinc status suppressed MNU-induced rat mammary tumorigenesis.

The objectives of this study were (i) to investigate the modulating effects of zinc nutrition on histochemically reactive zinc in the rat intestine and liver and (ii) to assess the relationship between histochemically reactive zinc and metallothionein-bound zinc in these tissues under varying zinc nutrition. Male Wistar rats were fed a zinc-deficient (3 mg zinc/kg), adequate-zinc (30 mg zinc/kg, ad libitum or pair-fed), or zinc-supplemented (155 mg zinc/kg) diet for 2 or 6 weeks. Plasma N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide-reactive zinc reflected dietary zinc intake. Abundance of the intestine histochemically reactive zinc was correlated with dietary zinc intake after 2 weeks of dietary treatment. Dietary zinc intake had no effect on the abundance of the intestine histochemically reactive zinc after 6 weeks of dietary treatment and the hepatic histochemically reactive zinc after both 2 and 6 weeks of dietary treatment. This lack of effect of dietary zinc intake on the abundance of histochemically reactive zinc was associated with a higher level of metallothionein. The molecular-mass distribution profile revealed that N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide-reactive zinc and metallothionein-bound zinc represented two different, but interrelated, pools of zinc. Overall, these results suggested that the abundance of histochemically reactive zinc was homeostatically regulated, which was partially achieved through the regulation of metallothionein levels in rats.Key words: N-6-methoxy-para-toluenesulfonamide (TSQ), histochemically reactive zinc, plasma TSQ-reactive zinc, intestine, metallothionein, rats.

Today, vanadium compounds are frequently included in nutritional supplements and are also being developed for therapeutic use in diabetes mellitus. Previously, tissue uptake of vanadium from bis(maltolato)oxovanadium(IV) (BMOV) was shown to be increased compared to its uptake from vanadyl sulfate (VS). Our primary objective was to test the hypothesis that complexation increases vanadium uptake and that this effect is independent of oxidation state. A secondary objective was to compare the effects of vanadium complexation and oxidation state on tissue iron, copper, and zinc. Wistar rats were fed either ammonium metavanadate (AMV), VS, or BMOV (1.2 mM each in the drinking water). Tissue uptake of V following 12 wk of BMOV or AMV was higher than that from VS (p0.05). BMOV led to decreased tissue Zn and increased bone Fe content. The same three compounds were compared in a cellular model of absorption (Caco-2 cells). Vanadium uptake from VS was higher than that from BMOV or AMV at 10 min, but from BMOV (250 microM only, 60 min), uptake was far greater than from AMV or VS. These results show that neither complexation nor oxidation state alone are adequate predictors of relative absorption, tissue accumulation, or trace element interactions.

Zinc distribution is apparently altered in breast cancer patients. It is unclear if this apparent zinc redistribution is a consequence of altered zinc nutrition or tissue-specific response to breast cancer. Our objectives were to assess effects of N-methyl-N-nitrosourea-treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis on body zinc-distribution profile in rats and to assess effects of dietary zinc intake on the body zinc-distribution profile during N-methyl-N-nitrosourea treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats. Female Sprague-Dawley rats were assigned to zinc-deficient (3 mg/kg diet) or zinc-adequate (31 mg/kg diet) ad libitum or pair-fed group. Rats were sham treated or N-methyl-N-nitrosourea treated (50 mg/kg body weight; Experiment 1 or 40 mg/kg body weight; Experiment 2) (n = 6). In both experiments, the zinc concentration was significantly higher (6-19 times) in mammary tumor than in mammary gland. Tissue zinc concentration was essentially unaffected by N-methyl-N-nitrosourea treatment and tumor bearing, but was reduced by zinc deficiency in the bone, kidney, and liver. Overall, higher mammary tumor zinc concentration and absence of zinc redistribution during N-methyl-N-nitrosourea treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis, regardless of zinc intakes, indicates zinc accumulation in mammary tumors. Because zinc is essential for growth and cancer is characterized by uncontrolled growth, this zinc accumulation suggests an involvement of zinc in N-methyl-N-nitrosourea-induced rat mammary tumorigenesis.

The present study investigated the effect of dietary zinc deficiency during the developmental period on hepatic cytochrome P450 (CYP) expression in postpubertal male rats. Twenty-one-day-old weanling male Wistar rats were randomly assigned to one of the following dietary groups: zinc-adequate (31 mg zinc/kg diet); marginal zinc-deficient (3 mg zinc/kg diet); severe zinc-deficient (1 mg zinc/kg diet); or pair-fed control for either the marginal or severe zinc-deficient group. All rats were killed at 63 days of age. Compared with the corresponding pair-fed controls, marginal zinc deficiency decreased CYP2C11-mediated testosterone 2α- and 16α-hydroxylase activities by 43 and 42%, respectively, whereas severe zinc deficiency reduced each of these activities by approximately 60%. The decrease in CYP2C11 activity was accompanied by a reduction in CYP2C11 protein and mRNA levels, as assessed by immunoblot and reverse transcription-polymerase chain reaction (RT-PCR) assays, respectively. Additional RT-PCR analysis indicated that severe zinc deficiency decreased CYP3A2 and CYP3A18 mRNA levels by 49 and 43%, respectively, whereas it increased CYP2C12 (253%) and CYP3A9 (238%) mRNA expression. Plasma testosterone concentration was decreased by 67% in the marginal zinc-deficient group when compared with the corresponding pair-fed control group. By comparison, it was below the limit of quantification (0.2 ng/mL) in the severe zinc-deficient rats. Overall, these results indicate that dietary zinc deficiency during the developmental period feminized the hepatic gene expression of the sexually dimorphic CYP2C11, CYP3A2, CYP3A18, CYP2C12, and CYP3A9 in postpubertal male rats.

Numerous studies have shown that zinc nutrition influences the growth of several types of tumor. However, the influence of zinc nutrition on mammary tumorigenesis is not known. To study the effects of dietary zinc intake on N-methyl-N-nitrosourea (MNU)-induced mammary tumorigenesis, female Sprague-Dawley rats were fed an egg-white-based diet providing 3 (Z3), 12 (Z12), or 31 (Z31) mg zinc/kg diet ad libitum. In addition, two pair-fed controls, PFZ12 and PFZ31, were also included. Fourteen weeks after MNU injection, cumulative tumor incidence and total number of tumors were lower in Z3 rats than in Z12 and Z31 rats. Cumulative tumor incidence and total number of tumors were lower in Z3 rats than in PFZ12 rats, but were the same as in PFZ31 rats. Cumulative tumor incidence and total number of tumors were also lower in pair-fed controls than in their corresponding ad libitum controls, but were the same between the ad libitum controls. Overall, the results showed that the effect of marginal zinc deficiency on MNU-induced mammary tumorigenesis in rats was primarily the result of a reduced feed intake associated with marginal zinc deficiency rather than zinc per se.

The objective of this study was to investigate if the altered 2E1 drug metabolism capacity in rats is due to effects of dietary zinc deficiency on the expression of hepatic microsomal P450 2E1 in rats. Rats were given free access to either a zinc-adequate diet or a zinc-deficient diet for 3 weeks. A second control group, the pair-fed control, was also included. At the end of the feeding trial, each dietary group was further divided into the drug control and ethanol treatment group. Ethanol (EtOH, 8 mL/kg body weight, intubation) was given once a day for 3 consecutive days. Within the drug control group, zinc deficiency reduced total P450 concentration but had no effect on the level of NADPH-P450 reductase, 2E1 protein, and 2E1 mRNA. 2E1 activity, which was indicated by N-nitrosodimethylamine demethylase (NDMA), aniline hydroxylase (AH), and p-nitrophenol hydroxylase (NH) activity, was also not affected by dietary zinc deficiency. The NDMA demethylase activity and 2E1 mRNA level were higher in zinc pair-fed rats than in zinc-adequate rats. EtOH treatment induced total P450 concentration in zinc-adequate and zinc-deficient rats and NADPH-P450 reductase level in zinc-adequate rats. 2E1 activity was induced by EtOH, while the level of 2E1 protein and 2E1 mRNA was unchanged. In conclusion, the constitutive expression of hepatic microsomal 2E1 was not affected by zinc deficiency per se; however, the prolonged severe depression of feed intake caused by zinc deficiency increased the level of 2E1 mRNA in rats. An acute EtOH treatment markedly induced 2E1 activity without affecting the level of 2E1 protein and mRNA. (J. Nutr. Biochem. 5:308–313, 1994.)

Lipopolysaccharide (LPS) triggers a global activation of Inflammatory responses leading to liver injury in humans. Zinc pretreatment has been shown to prevent LPS-induced hepatic necrosis. In North America, suboptimal zinc status is more common than once realized. However, the effect of inadequate zinc nutrition on the host's susceptibility to LPS-induced liver injury is not known. The objective of this study was to determine whether marginal zinc deficiency would render rats more susceptible to LPS-induced liver injury. Weanling Sprague-Dawley rats were assigned to one of three dietary treatment groups: marginally low zinc ad libitum (Z3; 3 mg zinc/kg diet), adequate zinc ad libitum (Z30; 30 mg zinc/kg diet), or adequate zinc pair-fed (Z30P) group. After 6 weeks, each dietary treatment group was further divided into LPS-control (saline) groups (C-Z3, C-Z30P, C-Z30) and LPS-treatment (1 mg/kg body weight, intraperitoneal, 8 hrs) groups (LPS-Z3, LPS-Z30P, LPS-Z30). LPS reduced the serum zinc concentration and increased the liver zinc concentration regardless of dietary zinc intake. Serum alanine aminotransferase level was higher in the LPS-Z3 rats than in the LPS-Z30P and LPS-Z30 rats. LPS also induced hepatocyte necrosis and neutrophil infiltration into the liver sinusoids. This LPS-induced liver damage was more severe in the LPS-Z3 rats than in the LPS-Z30P and LPS-Z30 rats. Together these findings have demonstrated that marginal zinc deficiency increased the susceptibility to LPS-induced liver injury in rats. These results indicate that patients with sepsis who have suboptimal zinc nutrition status may be at higher risk of developing greater liver damage.

Increasing evidence shows that labile intracellular zinc is metabolically important. Depletion of labile intracellular zinc using chelators suppresses DNA synthesis. In this study, we tested the hypothesis that labile intracellular zinc could be modulated via varying zinc nutrition. This could result in an altered availability of labile intracellular zinc, which, in turn, could influence zinc-dependent cellular events involved in cell proliferation and ultimately suppress growth. Labile intracellular zinc was detected by using N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), a membrane-permeable fluorescence probe. After 48 h culture in a zinc-depleted medium, labile intracellular zinc in 3T3 cells was diminished along with a suppressed DNA synthesis and cell proliferation. In contrast, supplementation of zinc to the zinc-depleted medium increased the labile intracellular zinc and promoted DNA synthesis and cell proliferation. Furthermore, growth factor-dependent stimulation of DNA synthesis and cell proliferation was also accompanied by increased labile intracellular zinc. Together, our data showed an association between the labile intracellular zinc, detected using TSQ, and 3T3 cell growth, suggesting that labile intracellular zinc could be an important cellular link between zinc nutrition and growth.