ABSTRACT
Concerns regarding excessive hepatic copper concentrations in dairy cows have increased. The objective of this study was to determine the association of hepatic copper concentrations with evidence of liver disease. Blood and liver samples were collected at the time of slaughter in cull dairy cows (n=100). Liver samples were analyzed for copper using inductively coupled plasma mass spectrometry and crude fat using liquid-liquid extraction and gravimetry. Serum samples were analyzed for glutamate dehydrogenase, γ-glutamyltransferase, sorbitol dehydrogenase, aspartate aminotransferase activities, and bile acid concentrations. Liver samples were examined histologically for inflammation, fibrosis, and rhodanine staining. Animals were stratified by hepatic copper concentration and samples in the highest and lowest quintiles (Q5 and Q1) were evaluated for oxidative stress. Systemic indices of oxidative stress included serum reactive oxygen and nitrogen species (RONS) and total antioxidant potential (AOP). Tissue-level oxidative stress was assessed by immunohistochemistry using 4-hydroxynonenal (4HNE) and 3-nitrotyrosine (3NIT) stains to score the relative abundance and distribution of oxidized lipid and protein products, respectively. Mean hepatic copper concentration was 496.83 µg/g and median 469.72 µg/g and ranged from 70.56 to 1264.27 µg/g dry tissue. No association was found between hepatic copper concentrations and clinicopathological or histological evidence of hepatic damage or dysfunction. There was a significant increase in the amount of IHC staining of 4HNE and 3NIT in Q5 compared with Q1. Moreover, the IHC staining mirrored the distribution of the copper-specific stain rhodanine. These results demonstrate that cows with elevated hepatic copper concentrations had no evidence of active liver disease but had increased hepatic oxidative stress.
ABSTRACT
PURPOSE: Although Cu complexes have been investigated as anticancer agents, there has been no description of Cu itself as a cancer killing agent. A stealth liposomal Cu formulation (LpCu) was studied in vitro and in vivo. METHODS: LpCu was evaluated in prostate cancer origin PC-3 cells by a metabolic cytotoxicity assay, by monitoring ROS, and by flow cytometry. LpCu efficacy was evaluated in vivo using intratumoral and intravenous injections into mice bearing PC-3 xenograft tumors. Toxicology was assessed by performing hematological and blood biochemistry assays, and tissue histology and Cu distribution was investigated by elemental analysis. RESULTS: LpCu and free Cu salts displayed similar levels of cell metabolic toxicity and ROS. Flow cytometry indicated that the mechanisms of cell death were both apoptosis and necrosis. Animals injected i.t. with 3.5 mg/kg or i.v. with 3.5 and 7.0 mg/kg LpCu exhibited significant tumor growth inhibition. Kidney and eye were the main organs affected by Cu-mediated toxicities, but spleen and liver were the major organs of Cu deposition. CONCLUSIONS: LpCu was effective at reducing tumor burden in the xenograft prostate cancer model. There was histological evidence of Cu toxicity in kidneys and eyes of animals treated at the maximum tolerated dose of LpCu 7.0 mg/kg.
