Lead contamination in backyard chicken layer flocks in California.

Backyard layer chickens may be exposed to a variety of metals in the environment, including lead. The potential public health concerns associated with lead exposure prompted us to systematically screen liver samples from backyard layers submitted to the diagnostic laboratory to estimate the prevalence of lead exposure. Over a period of 1 y, we tested 1,476 chicken livers, of which 45 were found to have lead concentrations of 0.9-41 µg/g. The lead-positive cases were investigated by follow-up questions to the bird owners on the environment, general management of the flock, and egg consumption of family members. Lead concentrations in 14 pooled egg samples were determined, and a conservative estimate of daily exposure of family members to lead was made based on egg consumption. In some cases, estimated daily lead intake exceeded the recommended limits for lead consumption in children. Analysis of feed, water, and environmental samples did not identify a source of exposure in most cases. Only 34% of owners of lead-positive birds submitted eggs or environmental samples, indicating a lack of interest or financial concerns. In most cases, neither the case history nor postmortem findings were indicative of lead intoxication; without systematically testing all birds, some cases could have been missed. Our study highlights the need for backyard chicken owners, veterinarians, and public health personnel to be aware of the risk of lead exposure and undertake preventive and surveillance measures.

Anabaena/Dolichospermum as the source of lethal microcystin levels responsible for a large cattle toxicosis event.

Thirty-two 14-month old steers died during a period of four days (19-23 June 2017) after drinking from Junipers Reservoir (southeastern Oregon, USA) during a cyanobacterial bloom. Clinical and histopathological findings were consistent with acute liver disease, and microcystin-LR was present at 3000 µg/L in a reservoir water sample and at 7100 µg/L in the rumen contents of one of the mortalities. Serum biochemistry and histological examination indicated severe liver damage consistent with microcystin toxicosis. Microscopic observation of reservoir water samples, limited to frozen or poorly stored and partially degraded samples, indicated the presence of abundant Anabaena/Dolichospermum, but the presence of other toxic cyanobacteria such as Microcystis could not be excluded. Metagenomic analysis showed the presence in these samples of a single cyanobacterium whose cpcBA, rpoB and rbcL genes indicated membership in the Anabaena/Dolichospermum genus. The sequence of a complete mcy gene cluster with homology to previously identified Anabaena mcy genes was recovered. These results emphasize the capacity for Anabaena/Dolichospermum blooms to produce lethal levels of microcystin, posing a danger to public health and livestock. Further, our findings indicate that such occurrences can occur outside the far-northern latitudes in which microcystin-producing Anabaena have typically been found.

Pathology of carbon monoxide poisoning in two cats.

BACKGROUND: Carbon monoxide (CO), a common cause of poisoning in human beings has also been implicated in the death of animals. Though there are multiple studies on CO poisoning and relevant lethal blood COHb concentrations in humans, there are no reliable reports of diagnostic lethal carboxyhemoglobin percentage of saturation (COHb%) in cats. Additionally, due to shared housing environments, exposures to companion animals can be a surrogate for lethal exposures in human beings and provide valuable information in concurrent forensic investigations. CASE PRESENTATION: Two adult Singapura brown ticked cats were submitted to the California Animal Health and Food Safety Laboratory (CAHFS) for necropsy and diagnostic work-up. These animals were found dead along with their two deceased owners. Similar lesions were observed in both cats. At necropsy, gross lesions consisted of multifocal, large, irregular, bright red spots on the skin of the abdomen and the inner surface of ear pinnae, bright red muscles and blood. The carcasses, and tissues fixed in formalin retained the bright red discoloration for up to two weeks. Microscopic lesions included diffuse pulmonary congestion and edema, and multifocal intense basophilia of cardiomyocytes mostly affecting whole fibers or occasionally a portion of the fiber. Based on the clinical history,gross and microscopic changes, cyanide or carbon monoxide poisoning was suspected. Blood samples analyzed for carbon monoxide showed 57 and 41% carboxyhemoglobin COHb%. Muscle samples were negative for cyanide. CONCLUSION: There are no established reference values for lethal COHb concentration in cats. The COHb % values detected in this case which fell within the lethal range reported for other species, along with the gross lesions and unique histological findings in the heart suggest a helpful criteria for diagnosis of CO intoxication associated death in cats. This case demonstrates that since pets share the same environment as human beings and often are a part of their activities, they can be useful adjuncts in potential forensic investigations to help solve human cases.

2-Deoxy-d-glucose Suppresses the In Vivo Antitumor Efficacy of Erlotinib in Head and Neck Squamous Cell Carcinoma Cells.

Poor tumor response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a significant challenge for effective treatment of head and neck squamous cell carcinoma (HNSCC). Therefore, strategies that may increase tumor response to EGFR TKIs are warranted in order to improve HNSCC patient treatment and overall survival. HNSCC tumors are highly glycolytic, and increased EGFR signaling has been found to promote glucose metabolism through various mechanisms. We have previously shown that inhibition of glycolysis with 2-deoxy-d-glucose (2DG) significantly enhanced the antitumor effects of cisplatin and radiation, which are commonly used to treat HNSCC. The goal of the current studies is to determine if 2DG will enhance the antitumor activity of the EGFR TKI erlotinib in HNSCC. Erlotinib transiently suppressed glucose consumption accompanied by alterations in pyruvate kinase M2 (PKM2) expression. 2DG enhanced the cytotoxic effect of erlotinib in vitro but reversed the antitumor effect of erlotinib in vivo. 2DG altered the N-glycosylation status of EGFR and induced the endoplasmic reticulum (ER) stress markers CHOP and BiP in vitro. Additionally, the effects of 2DG + erlotinib on cytotoxicity and ER stress in vitro were reversed by mannose but not glucose or antioxidant enzymes. Lastly, the protective effect of 2DG on erlotinib-induced cytotoxicity in vivo was reversed by chloroquine. Altogether, 2DG suppressed the antitumor efficacy of erlotinib in a HNSCC xenograft mouse model, which may be due to increased cytoprotective autophagy mediated by ER stress activation.

Systemic Akt1 Deletion after Tumor Onset in p53(-/-) Mice Increases Lifespan and Regresses Thymic Lymphoma Emulating p53 Restoration.

Akt is frequently activated in human cancers. However, it is unknown whether systemic inhibition of a single Akt isoform could regress cancer progression in cancers that are not driven by Akt activation. We systemically deleted Akt1 after tumor onset in p53(-/-) mice, which develop tumors independently of Akt activation. Systemic Akt1 deletion regresses thymic lymphoma in p53(-/-) mice emulating p53 restoration. Furthermore, pharmacological inhibition of Akt selectively kills thymic lymphoma cells and not primary thymocytes. Mechanistically, Akt1 inhibition in p53(-/-) thymic lymphoma inhibits Skp2 expression and induces FasL, which is the primary cause of cell death. Skp2 exerts resistance to cell death by antagonizing the induction of FasL and reducing FAS expression, which is linked to cyclin D1 expression. The results established a paradigm whereby systemic Akt1 inhibition is sufficient to regress tumors that are not driven by Akt activation and a mechanism of cell survival by Skp2.

EGFR inhibition induces proinflammatory cytokines via NOX4 in HNSCC.

UNLABELLED: Chronic inflammation plays a fundamental role in tumor promotion, migration, and invasion. With the use of microarray profiling, a profound increase was observed for those transcripts involved in proinflammatory signaling in epidermal growth factor receptor (EGFR) inhibitor-treated head and neck squamous cell carcinoma (HNSCC) cells as compared with their respective controls. As such, it was hypothesized that EGFR inhibitor efficacy is offset by the proinflammatory response that these therapeutics conjure in HNSCC. Systematic evaluation of the clinical EGFR inhibitors-erlotinib, cetuximab, lapatinib, and panitumumab-revealed increased secretion of proinflammatory cytokines such as interleukins (IL-2, IL-4, IL-6, IL-8), granulocyte-macrophage colony-stimulating factor, TNF-α, and IFN-γ. Mechanistic focus on IL-6 revealed that erlotinib induced a time-dependent increase in IL-6 mRNA and protein expression. Importantly, exogenous IL-6 protected HNSCC cells from erlotinib-induced cytotoxicity, whereas tocilizumab, an IL-6 receptor antagonist, sensitized cells to erlotinib in vitro and in vivo. Inhibitors of NF-κB, p38, and JNK suppressed erlotinib-induced IL-6 expression, suggesting critical roles for NF-κB and MAPK in IL-6 regulation. Furthermore, knockdown of NADPH oxidase 4 (NOX4) suppressed erlotinib-induced proinflammatory cytokine expression. Taken together, these results demonstrate that clinical EGFR inhibitors induce the expression of proinflammatory cytokines via NOX4. IMPLICATIONS: The antitumor activity of EGFR inhibitors is reduced by activation of NOX4-mediated proinflammatory pathways in HNSCC.

NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells.

Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formation in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect.

Susceptibility of human head and neck cancer cells to combined inhibition of glutathione and thioredoxin metabolism.

Increased glutathione (GSH) and thioredoxin (Trx) metabolism are mechanisms that are widely implicated in resistance of cancer cells to chemotherapy. The current study determined if simultaneous inhibition of GSH and Trx metabolism enhanced cell killing of human head and neck squamous cell carcinoma (HNSCC) cells by a mechanism involving oxidative stress. Inhibition of GSH and Trx metabolism with buthionine sulfoximine (BSO) and auranofin (AUR), respectively, induced significant decreases in clonogenic survival compared to either drug alone in FaDu, Cal-27 and SCC-25 HNSCC cells in vitro and in vivo in Cal-27 xenografts. BSO+AUR significantly increased glutathione and thioredoxin oxidation and suppressed peroxiredoxin activity in vitro. Pre-treatment with N-acetylcysteine completely reversed BSO+AUR-induced cell killing in FaDu and Cal-27 cells, while catalase and selenium supplementation only inhibited BSO+AUR-induced cell killing in FaDu cells. BSO+AUR decreased caspase 3/7 activity in HNSCC cells and significantly reduced the viability of both Bax/Bak double knockout (DKO) and DKO-Bax reconstituted hematopoietic cells suggesting that necrosis was involved. BSO+AUR also significantly sensitized FaDu, Cal-27, SCC-25 and SQ20B cells to cell killing induced by the EGFR inhibitor Erlotinib in vitro. These results support the conclusion that simultaneous inhibition of GSH and Trx metabolism pathways induces oxidative stress and clonogenic killing in HNSCCs and this strategy may be useful in sensitizing HNSCCs to EGFR inhibitors.

Erlotinib-mediated inhibition of EGFR signaling induces metabolic oxidative stress through NOX4.

Redox regulation of epidermal growth factor receptor (EGFR) signaling helps protect cells against oxidative stress. In this study, we investigated whether the cytotoxicity of an EGFR tyrosine kinase inhibitor, erlotinib (ERL), was mediated by induction of oxidative stress in human head and neck cancer (HNSCC) cells. ERL elicited cytotoxicity in vitro and in vivo while increasing a panel of oxidative stress parameters which were all reversible by the antioxidant N-acetyl cysteine. Knockdown of EGFR by using siRNA similarly increased these oxidative stress parameters. Overexpression of mitochondrial targeted catalase but not superoxide dismutase reversed ERL-induced cytotoxicity. Consistent with a general role for NADPH oxidase (NOX) enzymes in ERL-induced oxidative stress, ERL-induced cytotoxicity was reversed by diphenylene iodonium, a NOX complex inhibitor. ERL reduced the expression of NOX1, NOX2, and NOX5 but induced the expression of NOX4. Knockdown of NOX4 by using siRNA protected HNSCC cells from ERL-induced cytotoxicity and oxidative stress. Our findings support the concept that ERL-induced cytotoxicity is based on a specific mechanism of oxidative stress mediated by hydrogen peroxide production through NOX4 signaling.