Nickel-induced Toxicity Research Articles

Rab44 Deficiency Induces Impaired Immune Responses to Nickel Allergy.

Rab44 was recently identified as an atypical Rab GTPase that possesses EF-hand and coiled-coil domains at the N-terminus, and a Rab-GTPase domain at the C-terminus. Rab44 is highly expressed in immune-related cells such as mast cells, macrophages, osteoclasts, and granulocyte-lineage cells in the bone marrow. Therefore, it is speculated that Rab44 is involved in the inflammation and differentiation of immune cells. However, little is known about the role of Rab44 in inflammation. In this study, we showed that Rab44 was upregulated during the early phase of differentiation of M1- and M2-type macrophages. Rab44-deficient mice exhibited impaired tumor necrosis factor alpha and interleukin-10 production after lipopolysaccharide (LPS) stimulation. The number of granulocytes in Rab44-deficient mice was lower, but the lymphocyte count in Rab44-deficient mice was significantly higher than that in wild-type mice after LPS stimulation. Moreover, Rab44-deficient macrophages showed impaired nickel-induced toxicity, and Rab44-deficient mice showed impaired nickel-induced hypersensitivity. Upon nickel hypersensitivity induction, Rab44-deficient mice showed different frequencies of immune cells in the blood and ears. Thus, it is likely that Rab44 is implicated in immune cell differentiation and inflammation, and Rab44 deficiency induces impaired immune responses to nickel allergies.

Salicylic acid induced signaling against nickel-induced toxicity: Responses of growth, exopolysaccharides, phostosystem II photochemistry, nitrogen metabolism status and antioxidant system of Anabaena sp. PCC 7120

The Anabaena sp. PCC 7120 is a filamentous cyanobacterium which is well known for its nitrogen fixing ability but nickel (Ni2+) contamination (in water bodies) has a negative impact on its growth. The negative impact of Ni2+ can be ameliorated by calcium (Ca2+)-mediated nitric oxide (NO) signaling induced by salicylic acid (SA) treatment. The present study was performed to investigate the relation between SA and NO mediated by Ca2+ and to understand this interaction several inhibitors and scavengers were used in different combinations. Results showed that Ni2+- toxicity declined growth (measured in terms of dry weight; DW), contents of exopolysaccharide and photosynthetic pigments, and also negatively affected the PSII photochemistry (OJIP transient parameters: Fm, Fv, Fm/Fo, Fv/Fo, Ψo, ФPo, ФEo, Area, N, and PIABS were suppressed; while Fo, Fo/Fm, Fo/Fv, Sm, ФDo, ABS/RC, TRo/RC, ETo/RC and DIo/RC were raised) and nitrogen metabolism. However, supplementation of SA, Ca2+ (CaCl2) and sodium nitroprusside (SNP; donor of nitric oxide (NO) to the culture medium counteracted the negative impact of Ni2+ thereby improving the growth. Further, to investigate the relation between Ca2+and NO, when c-PTIO (2–4-carboxyphenyl- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; scavenger of NO) was supplemented to Ni+SA+Ca2+ and EGTA (ethyleneglycol-bis (β- aminoethyl)- N,N,N,N- tetraacetic acid; Ca2+chelator) to Ni+SA+NO treated culture medium, the recovery on above studied traits caused due to Ca2+ and NO was arrested even in presence of SA suggesting the signaling behavior of Ca2+ and NO in SA induced pathway. Moreover, the impact was worsened under c-PTIO supplemented Ni+SA+Ca2+than EGTA supplemented Ni+SA+NO treatment suggesting key role of Ca2+ in mediating NO signaling induced by SA. Conclusively, this is the first study that demonstrates the relation between SA and NO mediated by Ca2+in Ni2+-stressed Anabaena sp. PCC 7120.

Effects of short-term exposure to the heavy metal, nickel chloride (Nicl2) on gill histology and osmoregulation components of the gray mullet, Mugil cephalus

The gray mullet, Mugil cephalus is an inshore and bottom-feeding fish species of Oman sea. Therefore, the gray mullet may be more exposed to heavy metal contamination, as the toxic impacts of heavy metals mullet has been reported in various studies. This study was conducted to evaluate the toxic effects of the heavy metal, nickel (as NiCl2) on osmoregulation of the gray mullet by measuring blood biochemicals, hormones, minerals and gill histology. Fish (10 fish/tank) were experimentally exposed to NiCl2 at three environmentally relevant concentrations of 5, 10 and 15 μg/l for 96 h. Then, fish were challenged with seawater (35 mg/l) for a period of 120 min. The samples (blood and gill tissue) were collected After 96 exposure to NiCl2 and during salinity challenge (30, 60 and 120 min post challenge). The plasma levels of cortisol and glucose significantly increased in NiCl2−exposed fish. In addition, cortisol increased in all experimental groups 30 min after salinity challenge and then returned gradually to the same levels as the control at 120 min post salinity challenge (PSC). The triiodothyronine (T3) and thyroxine (T4) levels significantly decreased in response to 10 and 15 μg/l NiCl2. In all groups, the thyroid hormones significantly elevated at 30 min PSC. After 30 min PSC, T3 levels in all NiCl2−exposed fish and T4 in the treatment, 10 μg/l NiCl2 remained unchanged throughout the salinity challenge. In the treatment, 5 μg/l NiCl2, T4 levels were recovered at 120 min PSC and reached the same levels as the control. Exposure of fish to high concentrations of NiCl2 and salinity stress increased the lactate levels. However, lactate levels in 5 and 10 μg/l NiCl2 groups were recovered at 120 min PSC and reached the same levels as the control. Furthermore, plasma protein increased in response to 10 and 15 μg/l NiCl2. At 30 PSC, the protein levels decreased in control and 5 μg/l NiCl2 group, while it remained unchanged in fish exposed to 10 and 15 μg/l NiCl2 throughout the salinity challenge. Exposure of fish to NiCl2 disrupted the electrolyte (Na+, Cl−) balance both before and after salinity challenge, which may be due to gill lesions induced by the heavy metal and following alternations in gill permeability. However, fish in 5 μg/l NiCl2 re-established the ionic balance in the blood at the end of salinity challenge period. The malondialdehyde (MDA) levels significantly increased in response to 10 and 15 μg/l NiCl2. The MDA levels returned to the same levels as the control group at 120 min PSC. The results of the present study showed that nickel-induced toxicity (especially at high concentrations) can reduce the osmoregulation capabilities of mullet. However, fish are able to recover from the toxic effects over time, if contamination be eliminated.

Potential Protective Effect of Magnesium Supplementation on Nickel Induced Hepatotoxicity in Albino Wistar Rats

Nickel is considered one of the most harmful heavy metals on human health. This experimental study was designed to investigate whether magnesium supplementation has any influence on nickel-induced toxicity in albino rats. Twenty one adult rats were divided into three groups of seven animals each group. The first group was used as a control and received saline solution. The second group was administered with nickel sulfate (2.0 mg/100 g, i.p) and the third group was given both nickel sulfate (2.0 mg/100 g, i.p.) and magnesium sulfate (300 mg/kg, i.p) simultaneously on alternate days. Body weight gain was recorded regularly. After 21 consecutive days, cellular functions were evaluated by biochemical and oxidative stress markers. The nickel sulfate-treatment decreased body weight gain in rats. Nickel treatment produced also oxidative injury characterized by an increase in glucose level, enzymes markers activities, lipid profile and total proteins and bilirubin concentrations. Simultaneously, glutathione level (GSH) and catalase activity was diminished in liver tissue. These results are further substantiated with obviously changes in hepato- histoarchithecture. However, the treatment with magnesium ameliorated the previous parameters and resulted in an improvement of the histopathological lesions. Our results suggest that magnesium supplementation may be effective in reducing nickel oxidative damage.

Ethanol extract of Nigella sativa has antioxidant and ameliorative effect against nickel chloride-induced hepato-renal injury in rats

BackgroundNickel exposure causes hepato-renal toxicity via oxidative stress. Medicinal plants with antioxidants properties are being explored as treatment options. In this study, the effect of ethanol extract of Nigella sativa (ENS) on nickel chloride (NiCl2)-induced hepato-renal damage was evaluated by monitoring biochemical and oxidative stress markers. Additionally, the antioxidant capacity and phytochemical constituents of ENS were quantified using HPLC and GC-MS.ResultNiCl2 significantly increased (p < 0.05) aspartate aminotransferase, creatinine, sodium ion, chloride ion and malondialdehyde levels, while antioxidant enzymes were decreased in the organs except for kidney glutathione-S-transferase when compared to the control. However, ENS exerted inhibitory effect against NiCl2 toxicity in both organs by reversing the biomarkers towards control levels. ENS has a high antioxidant capacity and is rich in antioxidants including gallic acid, quercetin, eucalyptol and levomenthol that may have accounted for the improvement of hepato-renal health in co-exposed rats.ConclusionOur result suggests that amelioration of nickel chloride-induced hepato-renal pathology by ethanol extract of Nigella sativa was related to its antioxidant properties. Therefore, Nigella sativa could be valuable in the management of nickel-induced toxicity.

Nickel: Human Health and Environmental Toxicology.

Nickel is a transition element extensively distributed in the environment, air, water, and soil. It may derive from natural sources and anthropogenic activity. Although nickel is ubiquitous in the environment, its functional role as a trace element for animals and human beings has not been yet recognized. Environmental pollution from nickel may be due to industry, the use of liquid and solid fuels, as well as municipal and industrial waste. Nickel contact can cause a variety of side effects on human health, such as allergy, cardiovascular and kidney diseases, lung fibrosis, lung and nasal cancer. Although the molecular mechanisms of nickel-induced toxicity are not yet clear, mitochondrial dysfunctions and oxidative stress are thought to have a primary and crucial role in the toxicity of this metal. Recently, researchers, trying to characterize the capability of nickel to induce cancer, have found out that epigenetic alterations induced by nickel exposure can perturb the genome. The purpose of this review is to describe the chemical features of nickel in human beings and the mechanisms of its toxicity. Furthermore, the attention is focused on strategies to remove nickel from the environment, such as phytoremediation and phytomining.

Toksičnost organskog i neogranskog nikla u ćelijskim kulturama pankreasa - poređenje sa kadmijumom

Nickel compounds are Group 1 carcinogens and possibly cancer-causing in the pancreas. We examined the toxicity of nickel in both 2-D and 3-D pancreatic cell cultures, to determine the LD50 for organic and inorganic nickel in normal and cancerous cells. Assays with cadmium chloride were performed to be a comparison to potential nickel-induced toxicity. Cells were exposed to twelve concentrations of NiCl2 or Ni-(Ac)2 for 48h (2-D), or six concentrations for 48 hours (3-D). There was a significant (P=0.0016) difference between HPNE and AsPC-1 LD50 values after cadmium exposure, at 69.9 µM and 29.2 µM, respectively. Neither form of nickel exhibited toxicity in 2-D or 3-D cultures, but after 48h, changes in spheroid morphology were observed. The inability of Ni to reduce viable cell numbers suggests a toxic mechanism that differs from cadmium, also a Group 1 carcinogen. The cell microenvironment was not a factor in nickel toxicity with no changes in viable cells in either 2-D or 3-D cultures. These studies only examined cytotoxicity, and not genotoxicity, a potential mechanism of nickel carcinogenicity. Alterations in DNA function or the expression of apoptotic proteins/processes would take longer to manifest. Current work focuses on cellular changes following extended nickel exposure.

ASSESSMENT OF THE POTENTIAL ROLE OF L- METHIONINE ON NICKEL SULFATE INDUCED RENAL INJURY AND OXIDATIVE STRESS IN RAT

Objective: The study was designed to investigate the possible protective role of methionine (Met) in nickel sulfate induced oxidative stress in renal tissue.Methods: Rats were divided into the following groups: Healthy control; L-methionine (Met, 100 mg/kg, orally); nickel sulfate (Ni, 20 mg/kg, intraperitoneally); and combination Ni+Met. The experiment lasted 21 days. Antioxidant and renal function parameters with histological study were performed.Results: In the Ni group, marked renal damage was noticed with the significant elevation in the levels of creatinine, urea, and uric acid in serum. Animals also showed a significant rise in the lipid peroxidation level with a concomitant decrease in glutathione (GSH) and various antioxidant enzymes GSH peroxidase, catalase, and superoxide dismutase. Moreover, these changes in rats given the combined therapy nickel plus methionine were significantly less than those of group nickel, met prevented these alterations and maintained the antioxidant status. Histopathological examination of the kidney tissue proved the protective effect of Met against nickel toxicity.Conclusions: These results demonstrated that methionine augments antioxidant defense against nickel-induced toxicity and provides evidence that it has therapeutic potential as a renoprotective agent.

Proteomic Assessment of Biochemical Pathways That Are Critical to Nickel-Induced Toxicity Responses in Human Epithelial Cells.

Understanding the mechanisms underlying toxicity initiated by nickel, a ubiquitous environmental contaminant and known human carcinogen is necessary for proper assessment of its risks to human and environment. Among a variety of toxic mechanisms, disruption of protein responses and protein response-based biochemical pathways represents a key mechanism through which nickel induces cytotoxicity and carcinogenesis. To identify protein responses and biochemical pathways that are critical to nickel-induced toxicity responses, we measured cytotoxicity and changes in expression and phosphorylation status of 14 critical biochemical pathway regulators in human BEAS-2B cells exposed to four concentrations of nickel using an integrated proteomic approach. A subset of the pathway regulators, including interleukin-6, and JNK, were found to be linearly correlated with cell viability, and may function as molecular determinants of cytotoxic responses of BEAS-2B cells to nickel exposures. In addition, 128 differentially expressed proteins were identified by two dimensional electrophoresis (2-DE) and mass spectrometry. Principal component analysis, hierarchical cluster analyses, and ingenuity signaling pathway analysis (IPA) identified putative nickel toxicity pathways. Some of the proteins and pathways identified have not previously been linked to nickel toxicity. Based on the consistent results obtained from both ELISA and 2-DE proteomic analysis, we propose a core signaling pathway regulating cytotoxic responses of human BEAS-2B cells to nickel exposures, which integrates a small set of proteins involved in glycolysis and gluconeogenesis pathways, apoptosis, protein degradation, and stress responses including inflammation and oxidative stress.

Determining the nickel toxicity on chlorophyll content, Hill reaction and growth in maize seedlings

To assess nickel-induced toxicity in plants, Zea mays seeds were germinated and cultured on nutrient solution with nickel concentrations of 0, 50, 100 and 200 μM for a period of two weeks. Its effects on the growth, Hill reaction and photosynthetic pigments content were then investigated. The fresh and dry weight of leaves and roots increased in 50 μM nickel, but decreased in 100 and 200 μM. The decline in length of root and shoot were observed by increasing nickel concentration. According to the results, root and shoot showed differential growth response to various nickel concentrations. Nickel concentrations up to 100 μM caused increase in the content of chlorophyll a, but resulted in decrease at 200 μM nickel. No significant changes in chlorophyll b and carotenoids contents were observed. The rate of Hill reaction, as the ability of chlorophyll a in the reaction center of PSII680 to split water, decreased by increase in nickel concentration.

Nickel-induced cell death and survival pathways in cultured renal proximal tubule cells: roles of reactive oxygen species, ceramide and ABCB1

Nickel and nickel compounds are carcinogens that target the lungs and kidneys causing cell death or cell survival adaptation. The multidrug resistance P-glycoprotein ABCB1 protects cells against toxic metabolites and xenobiotics and is upregulated in many cancer cell types. Here, we investigated the role of ABCB1 in nickel-induced stress signaling mediated by reactive oxygen species (ROS) and ceramides. In renal proximal tubule cells, nickel chloride (0.1-0.25mM) increased both ROS formation, detected by 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and cellular ceramides, which were determined by lipid dot blot and surface immunostaining, culminating in decreased cell viability, increased DNA fragmentation, augmented PARP-1 cleavage, and increased ABCB1 mRNA and protein. Inhibitors of the de novo ceramide synthesis pathway (fumonisin B1, L-cycloserine) and an antioxidant (α-tocopherol) attenuated nickel-induced toxicity as well as induction of ABCB1. ABCB1 protects against nickel toxicity as PSC833, an ABCB1 blocker, augmented the decrease in cell viability by nickel. Moreover, nickel toxicity was attenuated in renal MDCK cells stably overexpressing ABCB1. In agreement with previous data that demonstrated extrusion of (glucosyl)ceramides by ABCB1 (Lee et al. in Toxicol Sci 121:343, 2011), PSC833 increased total cellular ceramides by >2-fold after nickel treatment. Further, glucosylceramide synthase (GCS) mRNA is upregulated by nickel at 3h by ~1.5-fold but declined with prolonged exposures (6-24h). Inhibition of GCS with C9DGJ or knockdown of GCS with siRNA significantly attenuated nickel toxicity. In conclusion, nickel induces a ROS-ceramide pathway to cause apoptotic cell death as well as activate adaptive survival responses, including upregulation of ABCB1, which improves cell survival by extruding proapoptotic (glucosyl)ceramides.

Identification of potential molecular biomarkers in response to thioredoxin reductase 1 deficiency under nickel exposure

Nickel (II) is a ubiquitous environmental contaminant and it is known to be a highly toxic metal. The level of nickel in the environment has been raised with advances in industrialization and the role of nickel in human diseases is of increasing concern. Thioredoxin reductase 1 (Trr 1) is one of major redox factors having a potential role in cellular defense system against exposure to environmental toxicants. In this study, we investigated the protective roles of the Trr 1 against nickel-induced DNA damage. We found significantly higher amounts of DNA strand break in Trr 1 silencing cells compared to Trr 1 wild-type cells under nickel exposure, using γ-H2AX immunofluorescence staining. We also identified the potential molecular biomarkers that participated in gene-environment interaction between Trr 1 deficiency and nickel exposure via microarray analysis. In particular, seven upregulated genes (AHNAK, FZR1, LGALS7, PLD1, PPM1F, RHOB and SFRP1) and three down-regulated genes (IFITM1, MAPK8 and RCN1), whose functions are principally in toxicity-prone as well as cytoprotection processes, including cell proliferation, cell survival, apoptosis, inflammation and DNA repair. Our findings demonstrate gene-environment interaction between Trr 1 deficiency and nickel-induced toxicity, as evidence that insufficient of redox factor Trr 1 accelerated DNA lesions caused by nickel exposure. These results suggest that the candidate genes might be further useful in the establishment of Trr 1-mediated strategies by which modulate cellular defense against environmental toxicants, nickel.

Exploring the molecular mechanisms of nickel-induced genotoxicity and carcinogenicity: a literature review.

Nickel, a naturally occurring element that exists in various mineral forms, is mainly found in soil and sediment, and its mobilization is influenced by the physicochemical properties of the soil. Industrial sources of nickel include metallurgical processes such as electroplating, alloy production, stainless steel, and nickel-cadmium batteries. Nickel industries, oil- and coal-burning power plants, and trash incinerators have been implicated in its release into the environment. In humans, nickel toxicity is influenced by the route of exposure, dose, and solubility of the nickel compound. Lung inhalation is the major route of exposure for nickel-induced toxicity. Nickel can also be ingested or absorbed through the skin. The primary target organs are the kidneys and lungs. Other organs such as the liver, spleen, heart, and testes can also be affected to a lesser extent. Although the most common health effect is an allergic reaction, research has also demonstrated that nickel is carcinogenic to humans. The focus of the present review is on recent research concerning the molecular mechanisms of nickel-induced genotoxicity and carcinogenicity. We first present a background on the occurrence of nickel in the environment, human exposure, and human health effects.

Hepatoprotective role of naringin on nickel-induced toxicity in male Wistar rats

Aim of the present study was planned to determine the protective role of naringin in attenuating the toxicity induced by nickel sulfate in rat liver. In this investigation nickel sulfate (20 mg/kg body weight) was administered intraperitoneally for 20 days to induce toxicity. Naringin was administered orally (20, 40 and 80 mg/kg body weight) for 20 days with intraperitoneal administration of nickel sulfate. Liver injury was measured by the increased activities of serum hepatic enzymes namely aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma glutamyl transferase, lactate dehydrogenase and total bilirubin along with increased elevation of lipid peroxidation markers, thiobarbituric reactive acid substances, lipid hydroperoxides, protein carbonyl content and conjugated dienes. The toxic effect of nickel was also indicated by significantly decreased activities of enzymatic antioxidants like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase and glucose-6-phosphate dehydrogenase and non-enzymatic antioxidants like reduced glutathione, total sulfhydryl groups, vitamin C and vitamin E levels were significantly decreased. Naringin administered at a dose of 80 mg/kg body weight significantly reversed the activities of hepatic marker enzymes, decreasing lipid peroxidative markers, increasing the antioxidant cascade and decreasing the nickel concentration in the liver. The effect at a dose of 80 mg/kg body weight was more pronounced than that of other two doses (20 and 40 mg/kg body weight). All these changes were supported by histopathological observations. These results clearly demonstrate that naringin has the potential in alleviating the toxic effects of nickel in rat liver.

Chelating efficacy of CaNa 2 EDTA on nickel-induced toxicity in Cirrhinus mrigala (Ham.) through its effects on glutathione peroxidase, reduced glutathione and lipid peroxidation

In this age of modern biology, aquatic toxicological research has provided potential tools for ecotoxicologic investigations. Heavy metals primarily affect protein structures and induce a stress in the organisms. The present investigation was carried out to assess the effect of nickel chloride on the selected organs of the freshwater fish Cirrhinus mrigala and how CaNa 2 EDTA counters its effects as an antidote. Toxicity experiments were conducted for different exposure periods and also in certain tissues namely gill, liver, kidney and muscle. The total protein content, reduced glutathione, glutathione peroxidase and lipid peroxidation were found to be decreased in the nickel chloride treated tissues and the treatment with CaNa 2 EDTA + nickel chloride returned to near normal levels. Histopathological observations also revealed that after the administration of nickel chloride + CaNa 2 EDTA the chelator induced reduction in nickel toxicity. It has also contributed towards reduction in the pathological damage, thus enabling the organs to attain their near normal histological appearance. The present study shown that CaNa 2 EDTA is an effective chelating agent for the removal of nickel and it has proved efficient in restoring both the biochemical variables and pathological features immediately after a sub lethal exposure of nickel chloride in fish. To cite this article: R. Gopal et al., C. R. Biologies 332 (2009).

Occupational Toxicology of Nickel and Nickel Compounds

Nickel and nickel compounds are widely used in industry. The high consumption of nickel products inevitably leads to occupational and environmental pollution. In occupational settings, exposure to nickel and nickel compounds occurs primarily during nickel refining, electroplating, and welding. The most common airborne exposures to nickel in the workplace are to insoluble nickel species, such as metallic nickel, nickel sulfide, and nickel oxides from dusts and fumes. The chemical and physical properties of nickel and nickel compounds strongly influence their bioavailability and toxicity. The lung and the skin are the principal target organs upon occupational exposure. inhalation exposure is a primary route for nickel-induced toxicity in the workplace. The most important adverse health effects due to occupational exposure to nickel and its compounds are skin allergies, lung fibrosis, and lung cancer. The exact mechanisms of nickel-induced carcinogenesis are not clear. This review summarizes the current knowledge on occupational toxicology of nickel and its compounds. The subtopics include: chemical and physical properties, uses, occupational exposures, occupational exposure limits, toxicokinetics, biological monitoring, acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, carcinogenicity, molecular mechanisms of carcinogenesis, and gaps in knowledge.

Nickel Carcinogenicity in Relation to the Health Risks from Residual Oil Fly Ash

ABSTRACT Epidemiological studies of workers in the nickel industry, animal exposure studies, and reports on the potential mechanisms of nickel-induced toxicity and carcinogenicity indicate that only crystalline sulfidic nickel compounds have been clearly established as carcinogenic or potentially carcinogenic in humans. This observation indicates the need to modify and update regulatory approaches for nickel to reflect noncancer toxicity values for some individual nickel species. Analysis of nickel compounds in residual oil fly ash (ROFA) indicates that sulfidic nickel compounds (e.g., nickel subsulfide, nickel sulfide) are not present. Thus, the potential for emission of carcinogenic nickel compounds from residual oil fly ash appears to be low. Preliminary reference concentrations (RfCs) for a number of nickel compounds, based on non-carcinogenic endpoints, are proposed on the basis of the benchmark dose approach in conjunction with NTP data for nickel species.

Interactions of orthopaedic metals with an immortalized rat osteoblast cell line

The toxicity of nickel, chromium (III) and (VI), vanadium and aluminium was compared in an immortalized neonatal rat osteoblast cell line using the MTT assay and a novel index of cytoxicity, alkaline phosphatase (ALP) activity. Where toxicity was observed, ALP was a consistently more sensitive detection method than the MTT assay. The toxicity of the metals increased in the order aluminium < chromium (III) < vanadium < nickel < chromium (VI). α-Tocopherol partially prevented nickel-induced toxicity (as assessed by ALP activity), whereas ascorbic acid had no protective effect. Chromium (VI) was more toxic than (III), with significant toxicity observed at 0.5 μ m. It is thought that Cr (III) cannot readily penetrate the cell membrane and this may account for the lower toxicity. Aluminium had a stimulatory effect on cell growth at low concentrations (0.5 μ m). The combination of immortalized rat osteoblasts and the ALP activity test provides a powerful tool for in vitro testing of orthopaedic materials.