Biochemical Mechanisms Of Toxicity Research Articles

Low concentrations of antimony impair adipogenesis and endoplasmic reticulum homeostasis during 3T3-L1 cells differentiation

Antimony (Sb) is a metalloid widely present in plastics used for food contact packaging, toys and other household items. Since Sb can be released by these plastics and come into contact with humans, health concerns have been highlighted. The effect of Sb on human tissues is yet controversial, and biochemical mechanisms of toxicity are lacking. In the present study, the effect of very low nanomolar concentrations of Sb(III), able to mimicking chronic human exposure, was evaluated in 3T3-L1 murine cells during the differentiation process. Low nanomolar Sb exposure (from 0.05 to 5 nM) induced lipid accumulation and a marked increase in C/EBP-β and PPAR-γ levels, the master regulators of adipogenesis. The Sb-induced PPAR-γ was reverted by the estrogen receptor antagonist ICI 182,780. Additionally, Sb stimulated preadipocytes proliferation inducing G2/M phase of cell cycle and this effect was associated to reduced cell-cycle inhibitor p21 levels. In addition to these metabolic dysfunctions, Sb activated the proinflammatory NF-κB pathway and altered endoplasmic reticulum (ER) homeostasis inducing ROS increase, ER stress markers XBP-1s and pEIF2a and downstream genes, such as Grp78 and CHOP. This study, for the first time, supports obesogenic effects of low concentrations exposure of Sb during preadipocytes differentiation.

Strobilurin fungicide kresoxim-methyl effects on a cancerous neural cell line: oxidant/antioxidant responses and in vitro migration

In this paper we report the effects of the complex III inhibitor, strobilurin fungicide kresoxim-methyl, on the cellular homeostasis of a mammalian cancerous neural cell line. We examined whether exposure to subcytotoxic concentrations of kresoxim-methyl induce cellular and biochemical mechanisms of toxicity on the murine neuroblastoma N2a cells. Results revealed elevation of mitochondrial superoxide generation, decrease in mitochondrial transmembrane potential, losses on GPx enzyme activity, along with increased nitrite release. Fungicide exposure also induced impaired cellular migration. Our findings suggest that kresoxim-methyl, besides targeting the mitochondria in fungi, exerts its mode of action in mammalian cancer cells.Abbreviations: CAT: catalase; DMEM: Dulbecco’s modified Eagle’s medium; GPx: glutathione peroxidase; KM: kresoxym-methyl; N2a: mouse neuroblastoma cells; NO: nitric oxide

Cuprizone Intoxication Induces Cell Intrinsic Alterations in Oligodendrocyte Metabolism Independent of Copper Chelation.

Cuprizone intoxication is a common animal model used to test myelin regenerative therapies for the treatment of diseases such as multiple sclerosis. Mice fed this copper chelator develop reversible, region-specific oligodendrocyte loss and demyelination. While the cellular changes influencing the demyelinating process have been explored in this model, there is no consensus about the biochemical mechanisms of toxicity in oligodendrocytes and about whether this damage arises from the chelation of copper in vivo. Here we have identified an oligodendroglial cell line that displays sensitivity to cuprizone toxicity and performed global metabolomic profiling to determine biochemical pathways altered by this treatment. We link these changes with alterations in brain metabolism in mice fed cuprizone for 2 and 6 weeks. We find that cuprizone induces widespread changes in one-carbon and amino acid metabolism as well as alterations in small molecules that are important for energy generation. We used mass spectrometry to examine chemical interactions that are important for copper chelation and toxicity. Our results indicate that cuprizone induces global perturbations in cellular metabolism that may be independent of its copper chelating ability and potentially related to its interactions with pyridoxal 5'-phosphate, a coenzyme essential for amino acid metabolism.

Alterations of cellular redox homeostasis in cultured fibroblast-like renal cells upon exposure to low doses of cytochrome bc1 complex inhibitor kresoxim-methyl

In this paper we elucidate the effects of the cytochrome bc1 inhibitor, strobilurin fungicide kresoxim-methyl, on the redox balance of a mammalian renal cell line. To explore whether mammalian exposure to sub-nephrotoxic concentrations of kresoxim-methyl induces cellular and biochemical mechanisms of toxicity, its effects on cellular viability and, in particular, several parameters related to oxidative stress, mitochondrial respiratory function and apoptosis were examined in fibroblast-like renal Vero cells. Elevation of mitochondrial superoxide generation, together with a concomitant decrease in mitochondrial transmembrane potential was indicative of mitochondrial dysfunction. Losses on antioxidant enzyme activities and GSH, along with increased H2O2 and nitrite release were associated with oxidative stress and induced impaired cellular migration. Raise of intracellular calcium was also observed, while no experimental evidence of apoptosis was found. Our findings suggest that sub-nephrotoxic concentrations of kresoxim-methyl cause perturbation of multiple pathways in renal mammalian cellular redox homeostasis.

Toxicity of nano- and micro-sized ZnO particles in human lung epithelial cells

This is the first comprehensive study to evaluate the cytotoxicity, biochemical mechanisms of toxicity, and oxidative DNA damage caused by exposing human bronchoalveolar carcinoma-derived cells (A549) to 70 and 420 nm ZnO particles. Particles of either size significantly reduced cell viability in a dose- and time-dependent manner within a rather narrow dosage range. Particle mass-based dosimetry and particle-specific surface area-based dosimetry yielded two distinct patterns of cytotoxicity in both 70 and 420 nm ZnO particles. Elevated levels of reactive oxygen species (ROS) resulted in intracellular oxidative stress, lipid peroxidation, cell membrane leakage, and oxidative DNA damage. The protective effect of N-acetylcysteine on ZnO-induced cytotoxicity further implicated oxidative stress in the cytotoxicity. Free Zn2+ and metal impurities were not major contributors of ROS induction as indicated by limited free Zn2+ cytotoxicity, extent of Zn2+ dissociation in the cell culture medium, and inductively-coupled plasma-mass spectrometry metal analysis. We conclude that (1) exposure to both sizes of ZnO particles leads to dose- and time-dependent cytotoxicity reflected in oxidative stress, lipid peroxidation, cell membrane damage, and oxidative DNA damage, (2) ZnO particles exhibit a much steeper dose–response pattern unseen in other metal oxides, and (3) neither free Zn2+ nor metal impurity in the ZnO particle samples is the cause of cytotoxicity.

Gene induction studies and toxicity of chemical mixtures.

As part of its mixtures program, the Agency for Toxic Substances and Disease Registry (ATSDR) supports in vitro and limited in vivo toxicity testing to further our understanding of the toxicity and health effects of chemical mixtures. There are increasing concerns that environmental chemicals adversely affect the health of humans and wildlife. These concerns have been augmented by the realization that exposure to chemicals often occurs to mixtures of these chemicals that may exhibit complex synergistic or antagonistic interactions. To address such concerns, we have conducted two studies with techniques that are being used increasingly in experimental toxicology. In the first study, six organochlorine pesticides (4,4 -DDT, 4,4 -DDD, 4,4 -DDE, aldrin, dieldrin, or endrin) were selected from the ATSDR Comprehensive Environmental Response, Compensation and Liability Act of 1980 (or Superfund) priority list and tested for their ability to modulate transcriptional activation of an estrogen-responsive reporter gene in transfected HeLa cells. In these assays, HeLa cells cotransfected with an expression vector encoding estrogen receptor and an estrogen-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid were dosed with and without selected environmental chemicals either individually or in defined combinations. Estradiol consistently elicited 10- to 23-fold dose-dependent inductions in this assay. By contrast, all six of the organochlorine pesticides showed no detectable dose-related response when tested either individually or in binary combinations. Thus, these chemicals as binary mixtures do not exhibit any additional estrogenicity at the levels tested in these assays. In the second study, arsenic [As(V)], cadmium [Cd(II)], chromium [Cr(III, VI)], and lead [Pb(II)] were tested in a commercially developed assay system, CAT-Tox (L), to identify metal-responsive promoters and to determine whether the pattern of gene expression changed with a mixture of these metals. This assay employs a battery of recombinant HepG2 cell lines to test the transcriptional activation capacity of xenobiotics in any of 13 different signal-transduction pathways. Singly, As(V), Cd(II), Cr(III, VI), and Pb(II) produced complex induction profiles in these assays. However, no evidence of synergistic activity was detected with a mixture of Cd(II), Cr(III), and Pb(II). These results have shown metal activation of gene expression through several previously unreported signal-transduction pathways and thus suggest new directions for future studies into their biochemical mechanisms of toxicity. In conclusion, the (italic)in vitro(/italic) methods used in these studies provide insights into complex interactions that occur in cellular systems and could be used to identify biomarkers of exposure to other environmental chemical mixtures.

Effects of Arsenic, Cadmium, Chromium, and Lead on Gene Expression Regulated by a Battery of 13 Different Promoters in Recombinant HepG2 Cells

Toxic metals occur naturally at low concentrations throughout the environment, but are found in higher concentrations at many of the hazardous waste sites on the EPA Superfund list. As part of the Agency for Toxic Substances and Disease Registry (ATSDR) mandate to evaluate the toxicity of metals and mixtures, we chose four of the high-priority metal pollutants from ATSDR's HAZDAT list, including arsenic, cadmium, chromium, and lead, to test in a commercially developed assay system, CAT-Tox(L) (Xenometrix). This assay employs a battery of recombinant HepG2 cell lines to test the transcriptional activation capacity of xenobiotics in any of 13 different signal transduction pathways. Our specific aims were to identify metal-responsive promoters and determine whether the pattern of gene expression changed with a mixture of metals. Humic acid was used in all assays as a carrier to help solubilize the metals and, in all cases, the cells were exposed to the humic acid–metal mixture for 48 h. Humic acid alone, at 50–100 μM, showed moderate activation of the XRE promoter, but little other notable activity. As(V), at doses of 50–250 μM, produced a complex profile of activity showing significant dose-dependent induction of the hMTIIA, GST Ya, HSP70, FOS, XRE, NFκBRE, GADD153, p53RE, and CRE promoters. Pb(II) showed dose-related induction of the GST Ya, XRE, hMTIIA, GRP78, and CYP IA1 promoters at doses in the range of 12–100 μM. Cd(II), at 1.25–15 μM, yielded significant dose-dependent induction of hMTIIA, XRE, CYP IA1, GST Ya, HSP70, NFκBRE, and FOS. Whereas Cr(III) yielded small, though significant inductions of the CRE, FOS, GADD153, and XRE promoters only at the highest dose (750 μM), Cr(VI) produced significant dose-related inductions of the p53RE, FOS, NFκBRE, XRE, GADD45, HSP70, and CRE promoters at much lower doses, in the range of 5–10 μM. Assays testing serial dilutions of a mixture comprising 7.5 μM Cd(II), 750 μM Cr(III), and 100 μM Pb(II) (the combination of metals most frequently found at National Priority List sites) showed significant dose-dependent induction of the hMTIIA promoter, but failed to show dose-related induction of any other promoter and showed no evidence of synergistic activation of gene expression by the metals in this mixture. Our results thus show metal activation of gene expression through several previously unreported signal transduction pathways, including As(V) induction of GST Ya, FOS, XRE, NFkBRE, GADD153, p53RE, and CRE; Pb(II) induction of GST Ya, XRE, Cyp IA1, and GADD153; Cd(II) induction of NFkBRE, Cyp IA1, XRE, and GST Ya; and Cr(VI) induction of p53RE, XRE, GADD45, HSP70, and CRE promoters, and thus suggest new insights into the biochemical mechanisms of toxicity and carcinogenicity of metals. It is also an important finding that no evidence of synergistic activity was detected with the mixture of Cd(II), Cr(III), and Pb(II) tested in these assays.

A UNIFIED APPROACH TO RISK CHARACTERIZATION

A fundamental issue of toxicology is to determine levels of human exposure to toxic substances, commonly referred to as acceptable daily intakes (ADIs) or reference doses (RfDs), that are presumed to cause no or negligible health risks. The goal is to determine relatively safe levels for human exposure to toxic substances using scientifically defensible methods and data, including a discussion of the imprecision of the process. Ideally, an understanding of the biochemical mechanisms of toxicity would allow the construction of biologically based dose-response models that are applicable across species in the range of human exposure levels. In the absence of such information, low-dose linear extrapolation to zero risk, without a threshold dose, generally is used by U.S. regulatory agencies to estimate cancer risk. For noncancer effects, the no-observed-adverseeffect level (NOAEL) is divided by safety (uncertainty) factors to arrive at an ADI (RfD) that is presumed to be below a threshold dose and to pose no risk, or at worst, negligible risk. The dichotomy of approaches for cancer and noncancer risk assessment based on the threshold concept can be questioned. Animals and humans with no known exposure to a particular toxic substance sometimes exhibit an noncancer adverse health effect such as kidney failure or a defect at birth. Hence, other exogenous and/or endogenous exposures produce that adverse health effect, particularly in susceptible individuals. Thus, a threshold dose, if one exists, is already surpassed in a population. If a toxic substance contributes by the same pathway to the background dose, additional adverse effects are expected with no threshold for the added exposure. For cancer, threshold doses may exist for nongenotoxic (secondary mechanisms) modes of action. Examples of secondary mechanisms are the production of bladder cancer at high doses that produce

Chemical softness and acute metal toxicity in mice and Drosophila

Correlations reported by Jones and Vaughn (1978) between chemical softness parameters and LD50s for metal compounds in rodents have been confirmed and extended. In the present study 14-day LD50s for mice were determined under uniform experimental conditions for 24 metal salts injected ip. For certain ions the correlation between the LD50 and the Pearson and Mawby (1967) softness parameter, σ p is better than that obtained by Jones and Vaughn (1978) using data from diverse sources. Notably, however, we found no orrelation for the alkaline-earth metals either with σ p or with other softness parameters. In parallel experiments with the same metal compounds, we also found correlations between σ p and 4-day LC50s for Drosophila exposed to the salts in their food. For both the mouse and Drosophila, soft ions were generally found to be more toxic than hard ions such that their rankings are significantly correlated. Several differences in relative toxicities of the metal ions were found to occur between mice and Drosophila, and some interpretations of these findings are give. Correlations between toxicity and physicochemical parameters of toxic materials can offer insights into biochemical mechanisms of toxicity and may be useful in predicting toxicity.