Effects of long-term exposure to Cu²⁺ and Cd²⁺ on the pentose phosphate pathway dehydrogenase activities in the ovary of adult Bufo arenarum: possible role as biomarker for Cu²⁺ toxicity

Abstract

The effects of copper and cadmium on metabolism through the pentose phosphate pathway were evaluated in Bufo arenarum toad ovary. The effects of the two metals on dehydrogenases from this pathway were evaluated by three experiments: (1) in samples obtained from control females with addition of the metals to the reaction mixture (in vitro), (2) in samples obtained from control females and after long-term exposure of females to 4 and 100 μg/L of Cu or Cd in the incubation media (in vitro after exposure to the metals in vivo), and (3) ¹⁴CO₂ production through the pentose phosphate pathway was evaluated after [U-¹⁴C]glucose microinjection on ovulated oocytes (in vivo after microinjection of the metals). Results from (1) evidenced inhibition of both enzyme activities but only above 1.5 mM Cu and Cd added to the reaction mixture. In (2) both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities decreased in samples from the ovaries of females exposed in vivo to Cu, in a concentration-dependent manner (up to 90% in females exposed to 100 μg/L Cu: 2.12±1.57 NADPH μmol/min μg protein×10⁻⁵ vs 19.97±8.54 in control females). Cd treatment of the toads only rendered an inhibitory effect on 6-phosphogluconate dehydrogenase activity after exposure to 4 μg/L of the bivalent cation. (3) In vivo ¹⁴CO₂ evolution significantly decreased in oocytes coinjected with 6.3×10⁻³ mM Cu (calculated intracellular final concentration of the metal injected) and radioactive glucose. Cu and Cd concentration in samples from exposed females were always under detection limit by particle-induced X-ray emission. The results presented here are in agreement with a role for both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities determination as biomarkers of effect and exposure for Cu but not for Cd toxicity.

Introduction

Several authors have described an important role of the carbohydrates metabolism through the pentose phosphate pathway on the metabolism of ovarian oocytes from different species. Bilinkis et al. (1991) described an increase in glucose 6-phosphate dehydrogenase activity during salamander Pleurodeles waltl oocytes in vivo maturation, strongly related to the oocyte capacity for activation. Also, Ferrandi et al. (1993) reported that the ability of bovine oocytes to utilize glucose is increased at the time of maturation. Budeguer de Atenor et al. (1989) found that carbohydrates are used in Bufo arenarum oocytes mainly through the pentose phosphate pathway and the glutamic–aspartic cycle during the breeding season. Radojkovic and Ureta (1982) demonstrated that the pentose phosphate pathway and glycogen synthesis are the main operative pathways for glucose utilization in oocytes from the frog Calyptocephalella caudiverbera.

Downs et al. (1998) proposed that glucose metabolism through the pentose phosphate pathway is important in the meiotic induction mechanism and may involve the generation of phosphoribosil pyrophosphates that act, at least in part, through the purine metabolizing pathways. Also, the generation of NADPH as a consequence of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activity, may have an important role in oocyte maturation, since reducing power is necessary for lipid and steroid biosynthesis.

B. arenarum embryos and adult toads have recently been described as potent bioindicators of environmental pollution and are widely used in our country to perform various bioassays (Herkovits and Pérez-Coll, 1999; Herkovits et al (1996), Herkovits et al (2002)). At the molecular level, metallothioneins, small cytosolic proteins involved in the homeostatic control of Cu and Zn and in the detoxification of xenobiotic metals such as Cd and Hg also found in Bufo arenarum, were identified as good biomarkers of exposure to metals (Perez-Coll et al (1997), Perez-Coll et al (1999)).

Heavy metals are known to produce several health disorders in different animals. Acute toxicity as well as teratogenesis by these toxicants during early life stages have been extensively studied in B. arenarum embryos (Herkovits and Pérez-Coll, 2000; Herkovits et al., 1997; Pérez-Coll et al (1986), Pérez-Coll et al (1988)). Nevertheless, little attention has been paid to the deleterious effects on ovarian tissues of long-term exposure of adult animals to low concentrations of heavy metals, which may lead to impairment in the viability of their offspring. We have found that Zn exposure of B. arenarum females leads to decreased activity of glucose 6-phosphate dehydrogenase in the ovary, as well as to decreased viability of the embryos (Naab et al., 2001). We have also reported that a significant change in glucose 6-phosphate dehydrogenase activity in the ovary always occurred as a consequence of females exposure to Zn; even if accumulation of the metal in this organ was below detection limit (Fonovich de Schroeder et al., 2000; Naab et al., 2001). These findings prompted us to suggest that glucose 6-phosphate dehydrogenase activity could be used as a potential biomarker of effect and exposure to Zn.

The purpose of the present work was to study (1) the effects of Cu²⁺ and Cd²⁺ added to the reaction mixture on the activity of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase evaluated in vitro, (2) the effect of long-term exposures of B. arenarum adult females to Cu²⁺ and Cd²⁺ on the activity of both enzymes, also evaluated in vitro, and (3) the effects of the metals on in vivo ¹⁴CO₂ production through the pentose phosphate pathway enzymes. Evaluation of a possible role for these two enzymes as biomarkers of effect and exposure to other heavy metals is also discussed. The present investigations may constitute a useful tool most valuable in the field of ecotoxicology, as the experimental system responds to sublethal metal concentrations. Thus, it allows the detection of toxicant stress which considerably decreases the number of individuals in a population through the impairment of reproduction while the health of adult animals is apparently not affected.