How to resolve inconclusive predictions from defined approaches for skin sensitisation in OECD Guideline No. 497.

In June 2021 the Organisation for Economic Co-operation and Development published Guideline No. 497 on Defined Approaches for Skin Sensitisation (DASS GL). There are two DAs published, known as the 2o3 and the ITS. The 2o3 uses two concordant results from either the DPRA, KeratinoSens™, or the h-CLAT assays to predict hazard (sensitiser/non-sensitiser). The ITS applies a score to results from the DPRA, the h-CLAT and an in silico model to predict United Nations Globally Harmonized System (GHS) sub-categories (1A/1B/Not Classified). The ITS can use Derek Nexus as the in silico model (known as ITSv1) or use OECD QSAR Toolbox (known as ITSv2). As limitations of the individual in chemico/in vitro assays and in silico predictions are carried through to the DAs, inconclusive predictions are possible for chemicals with results in the borderline range, and chemicals with out of domain results. However, these inconclusive predictions can be resolved by applying a weight of evidence approach. Herein, four case studies are presented, each 'inconclusive' for skin sensitisation potential according to both DAs. A weight of evidence approach was applied to each using a robust scientific approach to provide a conclusive prediction, where possible, based on several additional, non-animal lines of evidence.

Oral cadmium in mice carrying 5 versus 2 copies of the Slc39a8 gene: comparison of uptake, distribution, metal content, and toxicity.

The highly conserved human and mouse SLC39A8 gene encodes the divalent cation/bicarbonate symporter ZIP8 expressed ubiquitously in most cell types. Our bacterial artificial chromosome-transgenic BTZIP8-3 line has 3 additional copies of the Slc39a8 gene in addition to its constitutive diploid pair found in wild-type (WT) mice. In liver, kidney, lung, testis, gastrointestinal tract, and brain, BTZIP8-3 mice are known to express ∼2.5 times greater amounts of ZIP8, compared with WT mice. Herein we administered cadmium chloride (CdCl2) in drinking water (100 mg/L through week 2, 200 mg/L through week 4, 400 mg/L through week 8, 800 mg/L through week 12, and 1600 mg/L through week 20, when the experiment was concluded). We postulated that Cd uptake and distribution--and, therefore, toxicity in certain tissues--would be enhanced in BTZIP8-3, compared with WT mice. BTZIP8-3 and WT groups ingested comparable amounts of Cd. Compared with WT, BTZIP8-3 mice showed tissue specific: increases in Cd, zinc, and manganese content and decreases in calcium content. Both Cd-exposed BTZIP8-3 and WT were similar in lower urinary pH; increased plasma alanine and aspartate aminotransferase activities; elevated iron and copper content in liver, kidney, lung, and testis; and higher blood urea nitrogen and kidney weight. Histological changes in liver, kidney, lung, and testis were minimal. In summary, at the daily oral Cd exposures chosen for this study, 5 versus 2 Slc39a8 gene copies result in no differences in Cd toxicity but do cause differences in tissue-specific content of Cd, zinc, manganese, calcium, iron, and copper.

Lipid metabolism and body composition in Gclm(-/-) mice.

In humans and experimental animals, high fat diets (HFD) are associated with risk factors for metabolic diseases, such as excessive weight gain and adiposity, insulin resistance and fatty liver. Mice lacking the glutamate-cysteine ligase modifier subunit gene (Gclm(-/-)) and deficient in glutathione (GSH), are resistant to HFD-mediated weight gain. Herein, we evaluated Gclm-associated regulation of energy metabolism, oxidative stress, and glucose and lipid homeostasis. C57BL/6J Gclm(-/-) mice and littermate wild-type (WT) controls received a normal diet or an HFD for 11 weeks. HFD-fed Gclm(-/-) mice did not display a decreased respiratory quotient, suggesting that they are unable to process lipid for metabolism. Although dietary energy consumption and intestinal lipid absorption were unchanged in Gclm(-/-) mice, feeding these mice an HFD did not produce excess body weight nor fat storage. Gclm(-/-) mice displayed higher basal metabolic rates resulting from higher activities of liver mitochondrial NADH-CoQ oxidoreductase, thus elevating respiration. Although Gclm(-/-) mice exhibited strong systemic and hepatic oxidative stress responses, HFD did not promote glucose intolerance or insulin resistance. Furthermore, HFD-fed Gclm(-/-) mice did not develop fatty liver, likely resulting from very low expression levels of genes encoding lipid metabolizing enzymes. We conclude that Gclm is involved in the regulation of basal metabolic rate and the metabolism of dietary lipid. Although Gclm(-/-) mice display a strong oxidative stress response, they are protected from HFD-induced excessive weight gain and adipose deposition, insulin resistance and steatosis.

Tetrahydroindenoindole inhibits the progression of diabetes in mice.

Diabetes is characterized by elevated fasting blood glucose (FBG) resulting from improper insulin regulation and/or insulin resistance. Herein we used female C57BL/6J mouse models for type 1 diabetes (streptozotocin [STZ] treatment) and type 2 diabetes (high-fat diet) to examine the ability of 4b,5,9b,10-tetrahydroindeno[1,2-b]indole (THII) to intervene in the progression of diabetes. THII (100 microM in drinking water) significantly diminished and partially reversed the increase in FBG levels produced by STZ. After 10 weeks on a high-fat diet, mice had normal FBG levels, but exhibited fasting hyperinsulemia and loss of glucose tolerance. THII significantly diminished these changes in glucose and insulin. In isolated liver mitochondria, THII inhibited succinate-dependent H(2)O(2) production, while in white adipose tissue, THII inhibited NADPH oxidase-mediated H(2)O(2) production and lipid peroxidation. Without intervention, such oxidative processes might otherwise promote diabetogenesis via inflammatory pathways. THII also increased O(2) consumption and lowered respiratory quotient (CO(2) produced/O(2) consumed) in vivo, indicating a greater utilization of fat for metabolic fuel. Increased metabolic utilization of fat correlated with a decrease in the rate of body weight gain in THII-treated mice fed the high-fat diet. We conclude that THII may retard the progression of diabetes via multiple pathways, including the inhibition of oxidative and inflammatory pathways.

Manganese accumulation in the mouse ear following systemic exposure.

There is evidence in human populations that exposure to manganese (Mn), or Mn in combination with excessive noise exposure, results in hearing loss. Quantitative reverse-transcriptase polymerase chain reaction revealed expression of the metal transporters DMT1, ZIP8, and ZIP14 in control mouse ears. ZIP8 is known to have a high affinity (K(m) = 2.2 microM) for Mn transport, and ZIP8 protein was localized to the blood vessels of the ear by immunohistochemistry. We treated mice (strains C57BL/6J and DBA/2J) with Mn (100 mg/kg MnCl(2), by subcutaneous injection, on three alternating days), and Mn was significantly elevated in the ears of the treated mice. Mn concentrations remained elevated over controls for at least 2 weeks after treatment. These studies demonstrate that metal transporters are present in the mouse ear and that Mn can accumulate in the ear following systemic exposure. Future studies should focus on whether Mn exposure is associated with hearing deficits.

Over-the-counter analgesics normalize blood glucose and body composition in mice fed a high fat diet.

Type 2 diabetes (noninsulin-dependent diabetes mellitus) develops from a pre-diabetic condition that is characterized by insulin resistance and glucose intolerance, and is exacerbated by obesity. In this study, we compared the ability of over-the-counter analgesic drugs (OTCAD) [acetaminophen (APAP); ibuprofen (IBU); naproxen (NAP); aspirin (ASA)], to protect against the development of a pre-diabetic state in mice fed a high fat diet. After 10 weeks on the high fat diet, mice had normal fasting blood glucose (FBG) levels, but exhibited impaired glucose tolerance. Treatment with 20 mg OTCADs/kg body weight improved glucose tolerance, with the order of efficacy, APAP=ASA>IBU, while NAP proved ineffective. Mice fed the high fat diet also exhibited increases in weight gain associated with an increase in body fat. OTCADs prevented in part this increase in body fat, in the order of efficacy, APAP=IBU>NAP=ASA. In isolated liver mitochondria, OTCADs inhibited succinate-dependent H2O2 production, while in white adipose tissue, APAP inhibited NADPH-oxidase mediated H2O2 production and lipid peroxidation. Thus, OTCADs diminish pro-oxidant processes that might otherwise exacerbate inflammation and a pre-diabetic state. We conclude that OTCADs, especially APAP and IBU, may be valuable tools to delay or prevent the development of type 2 diabetes from a pre-diabetic condition.

Acetaminophen normalizes glucose homeostasis in mouse models for diabetes.

Loss of pancreatic beta cell insulin secretion is the most important element in the progression of type 1 and type 2 diabetes. Since oxidative stress is involved in the progressive loss of beta cell function, we evaluated the potential for the over-the-counter analgesic drug and antioxidant, acetaminophen (APAP), to intervene in the diabetogenic process. We used mouse models for type 1 diabetes (streptozotocin) and type 2 diabetes (high-fat diet) to examine the ability of APAP to intervene in the progression of diabetes. In C57BL/6J mice, streptozotocin caused a dosage dependent increase in fasting blood glucose (FBG), from 100 to >600mg/dl. Daily APAP (20mg/kg BW, gastric gavage), significantly prevented and partially reversed the increase in FBG levels produced by streptozotocin. After 10 weeks on a high-fat diet, mice developed fasting hyperinsulemia and impaired glucose tolerance compared to animals fed a control diet. APAP largely prevented these changes in insulin and glucose tolerance. Furthermore, APAP prevented most of the increase in body fat in mice fed the high-fat diet. One protective mechanism for APAP is suggested by studies using isolated liver mitochondria, where low micromolar concentrations abolished the production of reactive oxygen that might otherwise contribute to the destruction of pancreatic beta-cells. These findings suggest that administration of APAP to mice, in a dosage used safely by humans, reduces the production of mitochondrial reactive oxygen and concomitantly prevents the development of type 1 and type 2 diabetes in established animal models.

Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase.

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione (GSH) biosynthesis pathway. This enzyme is a heterodimer, comprising a catalytic subunit (GCLC) and a regulatory subunit (GCLM). Although GCLC alone can catalyze the formation of l-gamma-glutamyl-l-cysteine, its binding with GCLM enhances the enzyme activity by lowering the K(m) for glutamate and ATP, and increasing the K(i) for GSH inhibition. To characterize the enzyme structure-function relationship, we investigated the heterodimer formation between GCLC and GCLM, in vivo using the yeast two-hybrid system, and in vitro using affinity chromatography. A strong and specific interaction between GCLC and GCLM was observed in both systems. Deletion analysis indicated that most regions, except a portion of the C-terminal region of GCLC and a portion of the N-terminal region of GCLM, are required for the interaction to occur. Point mutations of selected amino acids were also tested for the binding activity. The GCLC Cys248Ala/Cys249Ala and Pro158Leu mutations enzyme showed the same strength of binding to GCLM as did wild-type GCLC, yet the catalytic activity was dramatically decreased. The results suggest that the heterodimer formation may not be dependent on primary amino-acid sequence but, instead, involves a complex formation of the tertiary structure of both proteins.

Hepatocyte-specific Gclc deletion leads to rapid onset of steatosis with mitochondrial injury and liver failure.

UNLABELLED: Oxidative stress is considered to be a critical mediator in liver injury of various etiologies. Depletion of glutathione (GSH), the major antioxidant in liver, has been associated with numerous liver diseases. To explore the specific role of hepatic GSH in vivo, we targeted Gclc, a gene essential for GSH synthesis, so that it was flanked by loxP sites and used the albumin-cyclization recombination (Alb-Cre) transgene to disrupt the Gclc gene specifically in hepatocytes. Deletion within the Gclc gene neared completion by postnatal day (PND)14, and loss of GCLC protein was complete by PND21. Cellular GSH was progressively depleted between PND14 and PND28-although loss of mitochondrial GSH was less severe. Nevertheless, ultrastructural examination of liver revealed dramatic changes in mitochondrial morphology; these alterations were accompanied by striking decreases in mitochondrial function in vitro, cellular ATP, and a marked increase in lipid peroxidation. Plasma liver biochemistry tests from these mice were consistent with progressive severe parenchymal damage. Starting at PND21, livers from hepatocyte-specific Gclc knockout [Gclc(h/h)] mice showed histological features of hepatic steatosis; this included inflammation and hepatocyte death, which progressed in severity such that mice died at approximately 1 month of age due to complications from liver failure. CONCLUSION: GSH is essential for hepatic function and loss of hepatocyte GSH synthesis leads to steatosis with mitochondrial injury and hepatic failure.

Enhanced cadmium-induced testicular necrosis and renal proximal tubule damage caused by gene-dose increase in a Slc39a8-transgenic mouse line.

Resistance to cadmium (Cd)-induced testicular necrosis is an autosomal recessive trait defined as the Cdm locus. Using positional cloning, we previously identified the Slc39a8 (encoding an apical-surface ZIP8 transporter protein) as the gene most likely responsible for the phenotype. In situ hybridization revealed that endothelial cells of the testis vasculature express high ZIP8 levels in two sensitive inbred mouse strains and negligible amounts in two resistant strains. In the present study, we isolated a 168.7-kb bacterial artificial chromosome (BAC), carrying only the Slc39a8 gene, from a Cd-sensitive 129/SvJ BAC library and generated BAC-transgenic mice. The BTZIP8-3 line, having three copies of the 129/SvJ Slc39a8 gene inserted into the Cd-resistant C57BL/6J genome (having its normal two copies of the Slc39a8 gene), showed tissue-specific ZIP8 mRNA expression similar to wild-type mice, mainly in lung, testis, and kidney. The approximately 2.5-fold greater expression paralleled the fact that the BTZIP8-3 line has five copies, whereas wild-type mice have two copies, of the Slc39a8 gene. The ZIP8 mRNA and protein localized especially to endothelial cells of the testis vasculature in BTZIP8-3 mice. Cd treatment reversed Cd resistance (seen in nontransgenic littermates) to Cd sensitivity in BTZIP8-3 mice; reversal of the testicular necrosis phenotype confirms that Slc39a8 is unequivocally the Cdm locus. ZIP8 also localized specifically to the apical surface of proximal tubule cells in the BTZIP8-3 kidney. Cd treatment caused acute renal failure and signs of proximal tubular damage in the BTZIP8-3 but not nontransgenic littermates. BTZIP8-3 mice should be a useful model for studying Cd-induced disease in kidney.

Novel n-3 fatty acid oxidation products activate Nrf2 by destabilizing the association between Keap1 and Cullin3.

Consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can mitigate the progression of diseases in which oxidative stress represents a common underlying biochemical process. Nrf2-regulated gene expression regulates detoxification of reactive oxygen species. EPA and DHA were subjected to an in vitro free radical oxidation process that models in vivo conditions. Oxidized n-3 fatty acids reacted directly with the negative regulator of Nrf2, Keap1, initiating Keap1 dissociation with Cullin3, thereby inducing Nrf2-directed gene expression. Liquid chromatography-tandem mass spectrometry analyses of oxidized EPA demonstrated the presence of novel cyclopentenone-containing molecules termed J3-isoprostanes in vitro and in vivo and were shown to induce Nrf2-directed gene expression. These experiments provide a biochemical basis for the hypothesis that formation of J-ring compounds generated from oxidation of EPA and DHA in vivo can reach concentrations high enough to induce Nrf2-based cellular defense systems.

Glutamate cysteine ligase catalysis: dependence on ATP and modifier subunit for regulation of tissue glutathione levels.

Glutamate cysteine ligase (GCL), which synthesizes gamma-glutamyl-cysteine (gamma-GC), is the rate-limiting enzyme in GSH biosynthesis. gamma-GC may be produced by the catalytic subunit GCLC or by the holoenzyme (GCLholo), which comprises GCLC and the modifier subunit GCLM. The Gclm(-/-) knock-out mouse shows tissue levels of GSH that are between 9 and 40% of the Gclm(+/+) wild-type mouse. In the present study, we used recombinant GCLC and GCLM and Gclm(-/-) mice to examine the role of GCLM on gamma-GC synthesis by GCLholo. GCLM decreased the Km for ATP by approximately 6-fold and, similar to other species, decreased the Km for glutamate and increased the Ki for feedback inhibition by GSH. Furthermore, GCLM increased by 4.4-fold the Kcat for gamma-GC synthesis; this difference in catalytic efficiency of GCLholo versus GCLC allowed us to derive a mathematical relationship for gamma-GC production and to determine the relative levels of GCLholo and GCLC; in homogenates of brain, liver, and lung, the ratio of GCLC to GCLholo was 7.0, 2.0, and 3.5, respectively. In kidney, however, the relationship between GCLC and GCLholo was complicated. Kidney contains GCLholo, free GCLC, and free GCLM, and free GCLC in kidney cannot interact with GCLM. Taken together, we conclude that, in most tissues, GCLM is limiting, suggesting that an increase in GCLM alone would increase gamma-GC synthesis. On the other hand, our results from kidney suggest that gamma-GC synthesis may be controlled post-translationally.

Genetically altered mice to evaluate glutathione homeostasis in health and disease.

The tripeptide glutathione (GSH) is part of an integrated antioxidant system that protects cells and tissues from oxidative damage. Oxidative stress can result from exposure to excessive amounts of endogenous and exogenous electrophiles. Until recently, animal and cell model systems used to investigate the role of GSH in disease processes had employed chemical agents that deplete cellular GSH by inhibiting GSH synthesis or by reacting chemically with GSH. Such models have proven useful, but questions concerning nonspecific effects of such chemicals remain. Recently, our laboratories and others have developed mouse models with genetic deficiencies in enzymes of the GSH biosynthetic pathway. This review focuses on the regulation of GSH homeostasis and, specifically, the new GSH-deficient mouse models that have been developed. These models will improve our understanding of the role of GSH in animal and human diseases.

Cyp1a2 protects against reactive oxygen production in mouse liver microsomes.

H(2)O(2) production was evaluated in liver microsomes prepared from Cyp1a1/1a2(+/+) wild-type and Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice pretreated with 5 microg dioxin (TCDD)/kg body wt or vehicle alone. NADPH-dependent H(2)O(2) production in TCDD-induced microsomes from wild-type mice was about one-third of that in noninduced microsomes. In Cyp1a2(-/-) mice, H(2)O(2) production was the same for induced and noninduced microsomes, with levels significantly higher than those in wild-type mice. Cyp1a1(-/-) microsomes displayed markedly lower levels of H(2)O(2) production in both induced and noninduced microsomes, compared with those in wild-type and Cyp1a2(-/-) microsomes. The CYP1A2 inhibitor furafylline in vitro exacerbated microsomal H(2)O(2) production proportional to the degree of CYP1A2 inhibition, and the CYP2E1 inhibitor diethyldithiocarbamate decreased H(2)O(2) production proportional to the degree of CYP2E1 inhibition. Microsomal H(2)O(2) production was strongly correlated to NADPH-stimulated production of thiobarbituric acid-reactive substances, as well as to decreases in microsomal membrane polarization anisotropy, indicative of peroxidation of unsaturated membrane lipids. Our results suggest that possibly acting as an "electron sink," CYP1A2 might decrease CYP2E1-and CYP1A1-mediated H(2)O(2) production and oxidative stress. In this regard, CYP1A2 may be considered an antioxidant enzyme.

Uncoupling-mediated generation of reactive oxygen by halogenated aromatic hydrocarbons in mouse liver microsomes.

Studying liver microsomes from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced or vehicle-treated (noninduced) mice, we evaluated the in vitro effects of added chemicals on the production of reactive oxygen due to substrate/P450-mediated uncoupling. The catalase-inhibited NADPH-dependent H(2)O(2) production (luminol assay) was lower in induced than noninduced microsomes. The effects of adding chemicals (2.5 microM) in vitro could be divided into three categories: Group 1, highly halogenated and coplanar compounds that increased H(2)O(2) production at least 5-fold in induced, but not in noninduced, microsomes; Group 2, non-coplanar halogenated biphenyls that did not affect H(2)O(2) production; Group 3, minimally halogenated biphenyls and benzo[a]pyrene that decreased H(2)O(2) production. Molar consumption of NADPH and O(2) and molar H(2)O(2) production (o-dianisidine oxidation) revealed that Group 1 compounds mostly increased, Group 2 had no effect, and Group 3 decreased the H(2)O(2)/O(2) and H(2)O(2)/NADPH ratios. Microsomal lipid peroxidation (thiobarbituric acid-reactive substances) was proportional to H(2)O(2) production. Although TCDD induction decreased microsomal production of H(2)O(2), addition of Group 1 compounds to TCDD-induced microsomes in vitro stimulated the second-electron reduction of cytochrome P450 and subsequent release of H(2)O(2) production. This pathway is likely to contribute to the oxidative stress response and associated toxicity produced by many of these environmental chemicals.