Acetaminophen-induced hepatotoxicity in mice lacking inducible nitric oxide synthase activity.

We recently reported that nitrotyrosine and acetaminophen (APAP)-cysteine protein adducts colocalize in the hepatic centrilobular cells following a toxic dose of APAP to mice. Whereas APAP-adducts are formed by reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine, nitrotyrosine residues are formed by reaction of tyrosine with peroxynitrite. Peroxynitrite is formed from nitric oxide (NO) and superoxide. This manuscript examines APAP (300 mg/kg) hepatotoxicity in mice lacking inducible nitric oxide synthase activity (NOS2 null or knockout mice; C57BL/6-Nos2(tm1Lau)) and in the wildtype mice. In a time course the ALT levels in the exposed NOS2 null mice were approximately 50% of the wildtype mice; however, histological examination of liver sections indicated similar levels of centrilobular hepatic necrosis in both wild-type and NOS2 null mice. Serum nitrate plus nitrite levels (NO synthesis) were identical in saline-treated NOS2 null and wild-type mice (53 +/- 2 microM). APAP increased NO synthesis in wild-type mice only. The increases paralleled the increases in ALT levels with peak levels of serum nitrate plus nitrite at 6 h (168 +/- 27 microM). In wild-type mice hepatic tyrosine nitration was greatly increased relative to saline treated controls. Tyrosine nitration increased in NOS2 null mice also, but the increase was much less. APAP increased hepatic malonaldehyde levels (lipid peroxidation) in NOS2 null mice only. The results suggest the presence of multiple pathways to APAP-mediated hepatic necrosis, one via nitrotyrosine, as in the wild-type mice, and another that is not dependent upon inducible nitric oxide synthase activity, but which may involve increased superoxide.

Vitellogenin induction in painted turtle, Chrysemys picta, as a biomarker of exposure to environmental levels of estradiol.

Ponds within cattle farms often support turtle and fish populations and are impacted by manure runoff. Cattle excrete metabolized (glucuronide-conjugated) hormones in feces and urine into these ponds, and bacteria cleave the glucuronide metabolites to active steroids, which can be stable for several weeks in wastewater. The objectives of this study were to (1) assess levels of xenoestrogens found in ponds near livestock pastures; and (2) assess whether these levels of xenoestrogens induce vitellogenin (VTG) in painted turtles in the laboratory and field. We collected water twice, 6 weeks apart, and placed turtle traps weekly into two ponds, which receive runoff from beef cattle pastures, and into one pond with no cattle farm effluents. Water E(2) levels were analyzed using C(18) solid phase extraction disks and detected in a radioimmunoassay (RIA). Plasma was collected from painted turtles (Chrysemys picta) captured from these ponds and VTG levels were measured via enzyme linked immunosorbent assay (ELISA). Nine additional turtles were collected from a pond at the South Carolina Botanical Gardens, which receives no farm runoff, and were exposed in the laboratory to nominal concentrations of 0.15, 1.5, and 15 ng/l estradiol (static renewal) over a 28-day period, followed by 14 days in clean water. Plasma samples were taken weekly for VTG measurement via ELISA. Levels of free estradiol in the water column of farm ponds range from 0.05 to 1.80 ng/l, as measured by RIA, and up to 7.4 ng/l as measured by ER-beta binding affinity. This is similar to what has been reported in streams receiving sewage treatment works (STW) effluents. In the laboratory, plasma VTG in male painted turtles could not be induced even at the high E(2) dose (9.45 ng/l) after 28 days. In the field, VTG levels were induced only in females when compared with animals from the SC Botanical Gardens. Adult male turtles need to be primed with high doses of E(2) prior to being able to respond to exogenous E(2). Given that males would not typically be sensitized in the wild, environmentally relevant levels of E(2) may not be sufficient to affect them. However, higher VTG levels in females could potentially change their reproductive fitness by altering egg size or by shifting energy allocations away from other survival needs. Long-term studies are needed to study potential impacts of VTG induction on female turtle reproductive success.

Purification of vitellin from grass shrimp Palaemonetes pugio, generation of monoclonal antibodies, and validation for the detection of lipovitellin in Crustacea.

Much effort has been put into developing vitellogenin antibodies against a wide variety of aquatic vertebrate species to study potential estrogen or anti-estrogen endocrine disrupters. Little work has been done on endocrine disruption in aquatic invertebrates. Although some antibodies have been produced against blue crab and penaeid shrimp lipovitellin, they have only poor cross-reactivity with the important estuarine grass shrimp, Palaemonetes pugio. Vitellin was purified from eggs, monoclonal antibodies were produced using standard techniques, and hybridoma supernatants were screened by ELISA. Western blots were done using extracts from male and female grass shrimp to verify specificity of the monoclonal antibodies. Two low molecular mass bands in the range of 68-85 kD and two high molecular mass bands in the range of 190-221 kD were found. In addition to grass shrimp, several other crustacean species were screened and cross-reactivity found, including blue crab (Callinectes sapidus), mud crab (Rhithropanopeus harrisii), red swamp crayfish (Procambarus clarkii ) and Daphnia magna. To further investigate the use of the antibody, we performed a chronic 6-week pyrene exposure study. We found that vitellin was upregulated in females after 6 weeks and that this may be a protective measure against lipophilic xenobiotics.