High Dietary Copper Research Articles

Syntaxin 5 Is Required for Copper Homeostasis in Drosophila and Mammals

Copper is essential for aerobic life, but many aspects of its cellular uptake and distribution remain to be fully elucidated. A genome-wide screen for copper homeostasis genes in Drosophila melanogaster identified the SNARE gene Syntaxin 5 (Syx5) as playing an important role in copper regulation; flies heterozygous for a null mutation in Syx5 display increased tolerance to high dietary copper. The phenotype is shown here to be due to a decrease in copper accumulation, a mechanism also observed in both Drosophila and human cell lines. Studies in adult Drosophila tissue suggest that very low levels of Syx5 result in neuronal defects and lethality, and increased levels also generate neuronal defects. In contrast, mild suppression generates a phenotype typical of copper-deficiency in viable, fertile flies and is exacerbated by co-suppression of the copper uptake gene Ctr1A. Reduced copper uptake appears to be due to reduced levels at the plasma membrane of the copper uptake transporter, Ctr1. Thus Syx5 plays an essential role in copper homeostasis and is a candidate gene for copper-related disease in humans.

Effect of High Dietary Copper on Somatostatin and Growth Hormone-Releasing Hormone Levels in the Hypothalami of Growing Pigs

This experiment was conducted to examine the effect of dietary copper supplementation on somatostatin (SS) and growth hormone-releasing hormone (GHRH) mRNA expression levels in the hypothalami of growing pigs. A total of 45 crossbred pigs were randomly assigned to three groups of 15 pigs each; five replicates of three animals comprised each group. Pigs were allocated to diets that contained 5 mg/kg copper (control), 125 mg/kg copper sulfate, or 125 mg/kg copper methionine. At the end of the experiment, five pigs were selected at random from each group and slaughtered, and hypothalami were collected for determination of SS and GHRH mRNA expression levels. The results showed that the SS expression levels were lower and the GHRH levels were higher in pigs fed the diets with 125 mg/kg copper methionine (P<0.05) and 125 mg/kg copper sulfate (P<0.05), respectively, than in pigs fed the diet with 5 mg/kg copper. Furthermore, the relationship between SS mRNA and GHRH mRNA abundance had a significantly negative correlation (P<0.05). The data indicated that high dietary copper could enhance GHRH mRNA expression levels and suppress SS mRNA expression levels in the hypothalami of pigs. High lever dietary copper (125 mg/kg copper sulfate or 125 mg/kg copper methionine) increased pigs' growth performance and feed efficiency but had no significant effect on daily feed intake; 125 mg/kg copper sulfate or 125 mg/kg copper methionine at the same lever had no difference on growth promoting in pigs.

The Interlobular Distribution of Copper in the Liver of Beef Calves on a High-Copper Diet

The aims of the present study were 1) to evaluate the interlobular distribution of copper (Cu) in the liver of beef calves on a high-Cu diet, 2) to determine whether this distribution differs between Galician Blonds and Holstein Friesians, and 3) to determine whether in vivo needle biopsy provides an appropriate measure of overall hepatic Cu status. Liver biopsies were performed before slaughter on twenty-nine 10-month-old beef calves fed growing and finishing diets supplemented with 35 mg/kg of Cu sulfate (10 Galician Blonds, 9 Holstein Friesians, and 10 Galician Blond x Holstein Friesian crosses). At slaughter, samples taken from 6 regions of the liver (the internal and external faces of the right lobe; the left, caudate, and quadrate lobes; and the processus papillaris) were acid digested, and their Cu contents were determined by inductively coupled plasma atomic emission spectrometry. The highest Cu concentrations were found in the left lobe, followed by the processus papillaris, and the lowest Cu concentrations were found in the caudate and quadrate lobes. Different breeds differ in absolute hepatic Cu levels, but interlobular Cu distribution does not appear to depend on breed, at least when Galician Blonds are compared with Holstein Friesians. In vivo needle biopsy afforded accurate estimates of overall hepatic Cu status.

Effect of different levels of copper on growth performance and cecal ecosystem of newly weaned piglets

The current study aimed to investigate the effects of different levels of copper sulfate on the growth performance and cecal ecosystem in newly weaned piglets. One hundred piglets weaned at 28±2 d were randomly allocated to 4 treatments with 5 replicates of 5 piglets each. Piglets received for 28 d the base diet with i) no addition (control) or with copper addition (from copper sulfate) at ii) 100, iii) 175, and iv) 250 mg/kg−1. On day 21, twenty piglets were randomly selected (one from each replicate) to slaughter and investigate the population and diversity of cecal microorganisms. The results showed that the diets containing 175 and 250 mg/kg−1 copper improved the average daily gain (ADG) by 51% and 60% and decreased the feed to gain ratio (F/G) by 21% and 16%, respectively. Adding 175 or 250 mg/kg−1 copper improved crude protein, ether extract, calcium and phosphorus digestibility. Viable counts of Enterobacteriaceae and Lactobacilli in cecum tended to be reduced, while the concentrations of cecal volatile fatty acids (VFA) were increased in pigs fed diet supplemented as copper level increased. Polymerase chain reaction (PCR) results showed that adding 175 or 250 mg/kg−1 copper reduced the lactobacilli in cecum. Denaturing gradient gel electrophoresis (DGGE) maps showed that band numbers and intensity of cecal bacterial 16S rDNA decreased as the copper levels increased. The results suggested that the effects of high dietary copper on microflora and their activities and metabolic products might contribute to the intestinal health and result in improved growth performance.

Effect of high dietary copper on weight gain and neuropeptide Y level in the hypothalamus of pigs

An experiment was performed to examine the effect of dietary copper supplementation on weight gain, neuropeptide Y (NPY) concentration and NPY mRNA expression level in the hypothalamus of pigs. Forty-five crossbred pigs were randomly assigned to three groups of 15 pigs, each comprising five replicates of 3 animals. Pigs were allocated to diets that contained 10 mg/kg (as a control), 125 and 250 mg/kg copper as CuSO 4. Live weight gain and feed conversion efficiency was determined at the end of the experiment and five pigs, selected at random from each group, were slaughtered and the hypothalami collected for determination of NPY concentration and NPY mRNA expression level. The results showed that average daily gain (ADG) and average daily feed intake (ADFI) were higher and feed:gain (F:G) ratio was lower in pigs fed the diets with 125 and 250 mg/kg copper ( P<0.05), respectively, than in pigs fed a diet with 10 mg/kg copper, but that there was no statistically significant difference in growth performance between animals of the 125 mg/kg and the 250 mg/kg copper groups. Furthermore, pigs fed diets with 125 and 250 mg/kg copper had higher NPY concentrations and NPY mRNA expression levels in their hypothalamus than control animals. The data indicated that 125 and 250 mg/kg copper gave similar responses in terms of weight gain, whilst high dietary copper could enhance NPY concentration and NPY mRNA expression level in the hypothalamus of pigs. High dietary copper appears to increase feed intake and promote weight gain by enhancing NPY concentration and NPY mRNA expression level in the hypothalamus of pigs.

The effects of dietary selenium on the oxidative stress of grouper, Epinephelus malabaricus, fed high copper

A growth trial was conducted to investigate the effects of dietary selenium (Se) on the oxidative stress of grouper, Epinephelus malabaricus, fed high dietary copper (Cu). The basal diet with high Cu (20 mg Cu/kg) was supplemented with low (0 mg Se/kg, HCuLSe), adequate (0.8 mg Se/kg, HCuNSe) and 2× adequate (1.6 mg Se/kg, HCuHSe) of Se. Basal diets containing adequate Se (0.8 mg Se/kg) and adequate Cu (4 mg Cu/kg) were included as controls (NCuNSe). Four experimental diets each were fed to triplicate groups of grouper (initial weight: 9.79 ± 0.09 g) in a closed-recirculating rearing system for 8 weeks. Weight gain and feed efficiency of fish fed the NCuNSe diet was significantly ( P < 0.05) higher than fish fed the HCuLSe diet. Hepatic Se concentration was higher in fish fed the NCuNSe and HCuHSe diets than fish fed the HCuLSe and HCuNSe diets. Hepatic Cu concentration and thiobarbituric acid reactive substances (TBARS) value were highest in fish fed the HCuLSe and HCuNSe diets, followed by fish fed the HCuHSe diet, and lowest in fish fed the NCuNSe diet. Leukocyte respiratory burst activity and plasma total immunoglobulin concentration were highest in fish fed the NCuNSe diet, followed by fish fed the HCuHSe diet, and lowest in fish fed the HCuLSe diet. Plasma lysozyme activity was highest in fish fed the NCuNSe diet, followed by fish fed the HCuHSe diet, then fish fed the HCuNSe diet, and lowest in fish fed the HCuLSe diet. These results indicated that high Cu ingestion induced an oxidative stress in grouper and depressed their immune response. High dietary Se (2× adequate) supplementation reduced this oxidative stress and improved the immune response of the fish.

Assessment of zinc and copper status in weaned piglets in relation to dietary zinc and copper supply

The aim of the study was to determine the effect of weaning and the effect of increasing dietary zinc concentrations on the zinc and copper status of weaned piglets (study 1) and to study the effect of high concentrations of dietary zinc and/or copper on zinc and copper status of weaned piglets (study 2). Study 1 included 54 piglets (six litters of nine piglets). One piglet from every litter was killed 1 day before weaning. The remaining 48 piglets were allocated at weaning (28 days) to four dietary zinc treatments (100, 250, 1000 or 2500 ppm) and subsequently killed 1-2, 5-6 or 14-15 days after weaning. Study 2 included 48 piglets (six litters of eight piglets) allocated to four dietary treatments, consisting of low or high dietary zinc (100 or 2500 ppm) in combination with low or high dietary copper (20 or 175 ppm). All piglets in study 2 were killed 5-7 days after weaning. In both studies, the trace mineral status was assessed by zinc and copper concentrations and alkaline phosphatase (AP) activity in plasma and mucosal tissue. In study 2, lymphocyte metallothionein (MT) mRNA and intestinal mucosa MT mRNA concentrations were included as zinc status markers. The results showed that the zinc status, measured as zinc in plasma and mucosa, was not affected by weaning of the piglets. Plasma copper concentrations decreased during the first 2 weeks after weaning. High dietary copper concentrations did not affect the concentration of copper in plasma, but increased the concentration of copper in mucosa and the concentration of zinc in plasma. The dietary zinc treatments increased the zinc concentration in plasma as well as the zinc and MT mRNA concentration in mucosa. Lymphocyte MT mRNA concentrations did not reflect the differences in dietary zinc supplementation.

Dietary Copper and High Saturated and trans Fat Intakes Associated With Cognitive Decline

Evidence from prospective epidemiologic studies and animal models suggests that intakes of dietary fats and copper may be associated with neurodegenerative diseases. To examine whether high dietary copper intake is associated with increased cognitive decline among persons who also consume a diet high in saturated and trans fats. Community-based prospective study. Chicago, Ill. Patients Chicago residents 65 years and older. Cognitive function was assessed using 4 cognitive tests administered during in-home interviews at 3-year intervals for 6 years. Dietary assessment was performed with a food frequency questionnaire. Dietary intakes of copper and fats were related to change in global cognitive score (the mean of the 4 tests) among 3718 participants. Among persons whose diets were high in saturated and trans fats, higher copper intake was associated with a faster rate of cognitive decline. In multiple-adjusted mixed models, the difference in rates for persons in the highest (median, 2.75 mg/d) vs lowest (median, 0.88 mg/d) quintiles of total copper intake was -6.14 standardized units per year (P<.001) or the equivalent of 19 more years of age. There was also a marginally statistically significant association (P = .07) with the highest quintile of food intake of copper (median, 1.51 mg/d) and a strong dose-response association with higher copper dose in vitamin supplements. Copper intake was not associated with cognitive change among persons whose diets were not high in these fats. These data suggest that high dietary intake of copper in conjunction with a diet high in saturated and trans fats may be associated with accelerated cognitive decline.

Dietary Copper Enhances the Peripheral Myelinopathy Produced by Oral Pyrrolidine Dithiocarbamate

The neurotoxic hazard of a dithiocarbamate is influenced by route of exposure and acid stability of the dithiocarbamate. As an example, oral administration of the acid labile dithiocarbamate N,N-diethyldithiocarbamate (DEDC) causes a central-peripheral axonopathy thought to result from acid-promoted decomposition to CS2 in the stomach. In contrast, parenteral administration of DEDC, which bypasses the acidic environment of the stomach, causes a primary demyelination that is thought to be mediated through the intact parent dithiocarbamate. The relative acid stability of pyrrolidine dithiocarbamate (PDTC) suggests that a significant portion of a dose can be absorbed intact following oral exposure with the potential to produce a primary myelin injury. The present study was performed to characterize the neurotoxicity of PDTC and evaluate the possible role of copper in dithiocarbamate-mediated demyelination. Male Sprague Dawley rats were administered PDTC in drinking water and given either a normal- or high-copper diet for 18, 47, or 58 weeks. Examination of peripheral nerve by light microscopy and electron microscopy at the end of exposures revealed primary myelin lesions and axonal degeneration in the PDTC groups, with a significant increase in the severity of several lesions observed for the PDTC, high-copper group relative to the PDTC normal-copper diet. ICP-AES metal analysis determined that the PDTC groups had significantly increased brain copper, and at 58 weeks a significant increase in copper was seen in the sciatic nerve of PDTC high-copper animals relative to PDTC normal-copper diet animals. Although RP-HPLC analysis could not detect globin alkylaminocarbonyl cysteine modifications analogous to those seen with parenteral DEDC, LC/MS/MS identified (pyrrolidin-1-yl carbonyl)cysteine adducts on PDTC-exposed rat globin. These findings are consistent with previous studies supporting the ability of acid-stable dithiocarbamates to mediate myelin injury following oral exposure. The greater severity of lesions associated with dietary copper supplementation and elevated copper levels in nerve also suggests that perturbation of copper homeostasis may contribute to the development of myelin lesions.

High Dietary Iron and Copper and Risk of Colorectal Cancer: A Case-Control Study in Burgundy, France

: Several hypotheses have been proposed for colorectal carcinogenesis, including formation of free radicals. A case-control study compared nutrient intake in 171 colorectal cancer cases versus 309 general population controls, using a detailed face-to-face food history questionnaire. A food composition table enabled us to determine the mean composition of the diet in macro- and micronutrients. Dietary intakes were separately categorized into quartiles by gender. Logistic regression models were adjusted for age, sex, energy, exercise, and body mass index. High energy, copper, iron, and vitamin E intakes were associated with an overall increased risk of colorectal cancer. The odds ratios associated with the fourth quartile of intake were 2.3 (95% confidence interval, 1.3-4.0), 2.4 (1.3-4.6), 2.2 (1.1-4.7), and 1.8 (1.0-3.4) for energy, copper, iron, and vitamin E, respectively. There were no significant associations with dietary fiber, folate, calcium, or antioxidant vitamins other than vitamin E. These findings regarding iron and copper suggest that free radicals play an important role in colorectal carcinogenesis, while the findings regarding vitamin E are so far unexplained.

Influence of weaning and effect of post weaning dietary zinc and copper on electrophysiological response to glucose, theophylline and 5-HT in piglet small intestinal mucosa

This study aimed to examine how weaning and how dietary zinc and/or copper fed post weaning may affect the electrophysiological response to glucose and to chloride secretagogues in piglet small intestine in vitro. Study 1 included 54 piglets (six litters of nine piglets). One piglet from every litter was killed 1 day before weaning. The remaining 48 piglets were allocated at weaning (28 d) to four dietary zinc treatments and subsequently killed 1–2, 5–6 or 14–15 days after weaning. Study 2 included 48 piglets (six litters of eight piglets) allocated to four dietary treatments, consisting of high or low dietary zinc with or without high dietary copper. All piglets in study 2 were killed 5–7 days after weaning. The in vitro studies in Ussing chambers showed that weaning resulted in increased ileal glucose absorption as well as increased neuroendocrine-regulated (activated by 5-HT) and cAMP-dependent (activated by theophylline) chloride secretion. High zinc supplementation reduced the responses to 5-HT and theophylline. The study did not reveal any influence of copper on these parameters. It is concluded that the positive effect of zinc supplementation on diarrhoea in weaned piglets may be due to zinc reducing the intestinal mucosal susceptibility to secretagogues that activate chloride secretion.

Increase of dietary vitamin C improves haemocyte respiratory burst response and growth of juvenile grass shrimp,Penaeus monodon , fed with high dietary copper

Effects of dietary vitamin C (l-ascorbyl-2-monophosphate-Mg, C2MP-Mg) on growth, tissue copper (Cu) accumulation, and haemocyte superoxide anion production of juvenile grass shrimp, Penaeus monodon, fed with either adequate or high (8× adequate) dietary Cu were studied. Three experimental diets were used: basal diet supplemented with adequate levels of both C2MP-Mg (40mg kg diet−1) and Cu (20mg kg diet−1) (NC-NCu); basal diet supplemented with adequate C2MP-Mg and high Cu (8× adequate) (NC-HCu); and basal diet supplemented with high C2MP-Mg (5× adequate) and high Cu (HC-HCu). These were each fed to triplicate groups of shrimp (mean initial weight: 0·29±0·01g) for 8 weeks. Highest (P< 0·01) weight gain, feed efficiency (FE) and protein efficiency ratio (PER) were observed in shrimp fed NC-NCu diet, intermediate in shrimp fed HC-HCu diet, and lowest in shrimp fed NC-HCu diet. Cu concentrations in hepatopancreas, muscle and haemolymph were highest in shrimp fed NC-HCu diet, followed by shrimp fed HC-HCu diet, and lowest for shrimp fed NC-NCu diet. Survival, total haemocyte count (THC) and intracellular superoxide anion (O−2) production were higher in shrimp fed NC-NCu diet than shrimp fed NC-HCu diet, whereas hepatosomatic index (HSI) was higher in shrimp fed NC-HCu diet than shrimp fed NC-NCu diet. However, all these parameters were similar in shrimp fed NC-NCu diet and shrimp fed HC-HCu diet. These data suggest that increase of dietary vitamin C improved haemocyte respiratory burst response and growth and prevented tissue Cu accumulation in P. monodon fed with high dietary Cu.

The influence of dietary microbial phytase and copper on copper status in growing pigs

To determine the effect of supplemental dietary microbial phytase and copper on copper status of growing pigs a 3×3 factorial arrangement of treatments with 72 castrated crossbred pigs covering a body weight range from 23 to 55 kg was conducted. Three levels of phytase (0, 250 and 500 U/kg diet) and three levels of copper (20, 80 and 175 mg/kg diet) were added to diets based on barley and soya bean meal. The addition of microbial phytase to the diets caused a slight increase in copper requirement. Higher dietary copper could overcome the negative effects of phytase on copper availability. As at all dietary copper levels, the zinc content in bone (phalanx 1) increased when phytase was added to the diets; it was concluded that the increase in copper requirement was due to the releasing of zinc from the phytate complex and the forthcoming antagonistic effects of zinc on copper availability.

Excessive hepatic copper accumulation in jaundiced rats fed a high-copper diet.

The response of copper metabolism to dietary copper challenge was investigated in jaundiced rats with elevated plasma concentrations of conjugated bilirubin as a result of impaired canicular transport of bilirubin glucuronides. Control and jaundiced rats were fed purified diets with either normal (64 micromol Cu/kg) or high (640 micromol Cu/kg) concentration of added copper. Copper loading produced a greater increase in hepatic copper concentrations in the jaundiced than in control rats. The greater dietary-copper-induced increase in hepatic copper in the jaundiced rats can be explained by the observed smaller rise in biliary copper excretion and a greater efficiency of dietary copper absorption. In individual rats, there was a positive relationship between hepatic copper concentrations and biliary copper concentrations. It is suggested that not the transport of copper from liver cells to bile but that from plasma to bile is diminished in the jaundiced rats. The elevated plasma copper concentrations in the jaundiced rats may support this suggestion.

A factorial experimental design for investigation of effects of dietary lipid content and pro- and antioxidants on lipid composition in Atlantic salmon (Salmo salar) tissues and lipoproteins

Sixteen groups of post smolt, Atlantic salmon (Salmo salar) (initial weight 148 ± 17 g) were fed diets with different lipid content and composition of pro- and antioxidants (vitamin E, vitamin C, astaxanthin, Fe, Cu and Mn). The composition of the experimental diets was based on a multivariate reduced factorial design (RFD) (27–3) with either high (+1) or low (–1) level (within limits of toxicity and requirement) of each of the seven dietary variables. Lipid class and fatty acid (FA) composition was analysed in retina, very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL). Further FA composition was analysed in fillet, liver and plasma. High dietary lipid increased growth, feed efficiency, fillet lipid level and the amount of LDL and HDL in plasma, whereas high dietary copper and high dietary vitamin E decreased the amount of plasma LDL. High dietary vitamin E decreased the amount of lipid and protein in LDL, whereas high dietary lipid increased cholesterol and cholesterol ester levels in plasma and HDL and the amount of lipid in LDL. Dietary astaxanthin and manganese affected retina PE levels. The FA composition did not differ significantly between the 16 diets. Dietary FA composition was reflected in fillet FA composition and gradually less in VLDL, LDL, HDL, plasma, liver and retina FA composition. Dietary lipid content influenced FA composition to some extent in the analysed tissues, i.e. generally high dietary lipid content led to a decreased relative amount of saturated and monoene fatty acids and an increase of the relative amount of polyene fatty acids.

High dietary arsenic exacerbates copper deprivation in rats

High dietary arsenic exacerbates copper deprivation in rats

High dietary arsenic exacerbates copper deprivation in rats

High dietary arsenic exacerbates copper deprivation in rats

Self-selection of copper-containing diets by copper-deficient and overloaded rats

To determine the effect of copper status on the preference for copper-containing food, male Sprague–Dawley rats were weaned to a copper-deficient, copper-adequate or high-copper diet. Four weeks later, alterations in copper status were confirmed by measurement of liver copper concentrations. Rats ( n=10) were then given the choice between a copper-adequate or a copper-restricted diet of similar composition. Preference scores indicated that control rats preferred copper-deficient food. Preferences of rats on the high-copper diet were not different from control rats, suggesting that copper overload did not alter the pattern of selection. However, 7 of the 10 copper-deficient rats ate 80% or more of their intake as copper-adequate food resulting in preference scores that were significantly different from controls ( p<0.03). This alteration in preference was corrected within 1 day of access to copper-adequate food.

High dietary copper improves odor characteristics of swine waste.

We conducted two experiments to determine the effects of dietary copper concentration and source on odor characteristics of swine waste. In both experiments, 192 weanling gilts and barrows were allotted to 24 pens. Pens were randomly assigned to one of six dietary treatments, consisting of control (10 ppm Cu as cupric sulfate, CuSO4), 66 or 225 ppm Cu as CuSO4, or 33, 66, or 100 ppm Cu as cupric citrate (Cucitrate). An antibiotic was included in the diets for Exp. 1, but not Exp. 2. On d 28, fecal samples were randomly obtained from one pig per pen and stored at -20 degrees C until preparation and evaluation by an odor panel. The odor panel consisted of 10 individuals, and each panelist evaluated the odor intensity, irritation intensity, and odor quality of the samples. In Exp. 1, the odor and irritation intensity of the feces were lower (P < .05) from animals consuming diets containing 225 ppm Cu as CuSO4 and 66 or 100 ppm Cu as Cu-citrate compared to the control. The odor quality of the waste from animals consuming diets containing 225 ppm Cu as CuSO4 and 66 or 100 ppm Cu as Cu-citrate was improved (P < .05) compared to the 33 ppm Cu treatment. In Exp. 2, the odor intensity of the feces of pigs receiving diets supplemented with all concentrations of Cu-citrate was lower (P < .05) than that of feces from the control animals. Irritation intensity of the feces was not affected by treatment. Odor quality of waste of pigs supplemented with 225 ppm Cu from CuSO4 and all concentrations of Cu-citrate was improved (P < .05) compared to that of waste of the control pigs. Two gilts and two barrows from each nursery pen in Exp. 1 were continued through the growing-finishing phase on their respective experimental diets. The growing-finishing phase lasted 103 d, and fecal samples were randomly obtained from one pig per pen at the completion of the phase. During the growing-finishing phase, the odor intensity and the irritation intensity of the feces were lower (P < .05) from pigs supplemented with 66 and 225 ppm Cu as CuSO4 and 66 and 100 ppm Cu from Cu-citrate than from the control pigs. The odor quality of the waste was improved (P < .05) in all animals receiving supplemental Cu. These data indicate an improvement in odor characteristics of swine waste with the supplementation of Cu. In addition, lower concentrations of an organic nonsulfate Cu source resulted in similar odor characteristics of swine waste as 225 ppm CuSO4.

Regulation of mitochondrial cytochrome b mRNA by copper in cultured human hepatoma cells and rat liver.

Copper overload and deficiency are known to cause morphological and functional mitochondrial abnormalities. The reverse transcriptase-polymerase chain reaction (RT-PCR)-based method of differential display of mRNA was used to identify genes with altered expression in cultured human hepatoma cells (Hep G2) exposed to increasing concentrations of copper (0-100 microM, 24 h). Copper regulation of a cloned PCR product, identified as the gene for the mitochondrially encoded cytochrome b, was confirmed by Northern analysis and in situ hybridization. Copper toxicity increased cytochrome b mRNA abundance up to 3.6-fold, and copper chelation reduced it by 50%. Hepatic cytochrome b mRNA was also increased in rats fed a high-copper diet. Thapsigargin treatment resulted in a significant increase in cytochrome b mRNA, suggesting that an increase in intracellular calcium may be involved in the mechanism of copper action. Furthermore, although cyclohexamide (CHX) alone did not increase cytochrome b mRNA, the addition of CHX and copper resulted in a sixfold increase. These data suggest a role for cytochrome b in the response to increases or decreases in hepatic copper.