Experimental evaluation of vitellogenin as a predictive biomarker for reproductive disruption.

Vitellogenin (VTG) synthesis in male oviparous vertebrates is used as an indicator of environmental estrogen exposure, but the relationship between elevated VTG levels and the effects of environmental estrogens on reproductive success are poorly understood. To examine whether altered VTG expression predicts reproductive impairment, we exposed medaka (Oryzias latipes) for 2 or 8 weeks posthatch to 0, 0.5, 1.0, 2.5, and 7.5 ppb of the environmental estrogen o,p'-DDT. Fish were sampled 2, 4, and 8 weeks after hatch to examine VTG expression and gonad development. After exposure, fish were transferred to clean water, grown to sexual maturity, and placed in mating pairs. We collected eggs for 7 days and scored them for fecundity (number of eggs), fertility (percent fertilized), and hatching success (percent hatched). DDT had no effect on VTG expression after a 2-week exposure, whereas all doses induced VTG after 8 weeks. At both exposure durations, the highest doses of DDT caused a female-skewed sex ratio in adults. Gonadal feminization appeared to be progressive: some ovotestes were observed after 2- or 4-week exposure to the two highest doses, but the proportion of ovaries increased after 8 weeks. Both 2- and 8-week exposures significantly reduced fertility and hatching success at all doses, with lower doses having a greater effect after longer exposure. Fertility and hatching success were more sensitive to estrogenic disruption than were gonad differentiation and vitellogenin expression. We suggest that VTG expression may be interpreted as a warning of reproductive consequences, but absence of expression cannot be interpreted as absence of consequences.

Biochemical defense mechanisms against copper-induced oxidative damage in the blue crab, Callinectes sapidus.

The blue crab (Callinectes sapidus) has a very dynamic copper metabolism associated with the biosynthesis and degradation of its respiratory pigment hemocyanin. In this study we report on the cellular defense mechanisms used by the crab to protect itself from copper toxicity. Short-term copper-exposure studies, conducted by incubating hepatopancreas tissue explants in copper-containing medium, show that copper taken up by the cells during the first 60 min combines with low-molecular-weight copper complex(es), which include Cu(I)-glutathione. Thereafter, copper binds to newly synthesized metallothionein (MT), with a concomitant decrease in Cu(I)-glutathione. Copper does not displace zinc from the endogenous ZnMT pool. Long-term exposure by means of copper-rich diets results in the synthesis of two MT isoforms in the hepatopancreas: CuMT-I and CuMT-II (D. Schlenk and M. Brouwer, 1991, Aquat. Toxicol. 20, 25-34). Transfer of copper from Cu(I)-glutathione to apoMT-I and apoMT-II can be accomplished in vitro. Cu(I) binding by the two isoforms is very different. Cu(I) binds to apoMT-I in a strictly cooperative manner. No partially filled Cu(I)-thiolate clusters appear to be present. In contrast, the Cu(I)-thiolate clusters in MT-II are formed only after more than four Cu(I) ions are bound. Long-term copper exposure leads to increased activity of two antioxidant enzymes: glutathione peroxidase and manganese superoxide dismutase (SOD). No CuZnSOD is found. Activities of catalase and glutathione reductase and the intracellular levels of glutathione are unaffected by copper. The defense mechanisms are not entirely sufficient for preventing copper-induced oxidative damage. Levels of oxidized lipids are significantly higher in copper-exposed crabs, but oxidized protein levels are nearly the same.

The paradigm that all oxygen-respiring eukaryotes have cytosolic CuZn-superoxide dismutase and that Mn-superoxide dismutase is localized to the mitochondria does not apply to a large group of marine arthropods.

The enzyme superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide radical, is present in the cytosol and mitochondria of all oxygen-respiring eukaryotes. The cytosolic form contains copper and zinc (CuZnSOD), whereas the mitochondrial form contains manganese (MnSOD). The latter protein is synthesized in the cytosol as a MnSOD precursor, containing an N-terminal mitochondrial-targeting sequence. CuZnSOD is sensitive toward cyanide (CN) and hydrogen peroxide (H2O2), but MnSOD is not. Assays for SOD activity in cytosol from the hepatopancreas of the blue crab, Callinectes sapidus, showed the presence of a CN/H2O2-insensitive form of SOD. No CN/H2O2-sensitive CuZnSOD was found. This unexpected phenomenon was shown to occur in all decapod crustacea (crabs, lobsters, shrimp) examined. The cytosolic and mitochondrial SODs of C. sapidus were purified by means of ion-exchange, size-exclusion, and reverse-phase HPLC. The cytosolic SOD is a homodimeric protein, which exists in a monomer-dimer equilibrium (24 kDa left and right arrow 48 kDa). The protein contains approximately 1 Mn per subunit. No copper or zinc is present. Amino acid sequence analysis identified the novel cytosolic SOD as a MnSOD precursor with an abnormal mitochondrial-targeting sequence. The mitochondrial SOD of C. sapidus is similar to the MnSOD found in other eukaryotes. N-Terminal amino sequences of mitochondrial and cytosolic blue crab MnSOD differ in several positions. The MnSODs are thus encoded for by two different genes. The paradigm that all eukaryotes contain intracellular CuZnSOD and that MnSOD occurs exclusively in the mitochondria appears not to apply to a large group of marine arthropods.