Concentrations of nonprotein sulfhydryls in the Mediterranean gecko, Hemidactylus turcicus (Gekkonidae: Squamata).
Nonprotein sulfhydryls (NPSH), especially glutathione (GSH), are involved in numerous biochemical pathways in animals and plants (Hazelton and Lang, 1980, 1984; Earnshaw and Johnson, 1985; Farooqui et al., 1987). Net GSH tissue concentration is dependent upon its metabolism (turnover), transport, participation in physiologic functions, and excretion (Griffith and Meister, 1979). GSH provides cellular protection by assisting in the breakdown of peroxides and by reduction of disulfides. In addition, it forms conjugates with exogenous agents such as drugs and toxicants, aiding in their transport and excretion. Although the existence of GSH has been reported in many animals and plant species, little is known about the existence and role of GSH, or other sulfhydryls, in reptiles. Most reptiles are ectothermic heterotherms, that is, their metabolic rate is directly proportional to their body temperature. The purpose of this study was to determine the levels of hepatic NPSH including GSH in the Mediterranean gecko, Hemidactylus turcicus, and establish if there is an effect of the metabolic rate on GSH levels in geckos maintained at ambient and manipulated temperatures.
MATERIALS AND METHODS
Seventy-two Mediterranean geckos were collected in the vicinity of the University of Texas Pan American at Edinburg, Texas, in April. The average ambient temperature was about 86[degrees]F (30[degrees]C). Geckos were placed in plastic containers over night in shaded locations prior to the experiments. They then were divided into three groups of 24 geckos each with approximately equal number of males and females in each group. One group was used as a field control without any treatment. The other two groups were placed in a Sargent Welch Incubator (Sargent Welch Scientific Co., Irving, Texas) with a Tele-Thermometer Model 46 TUC (Yellow Spring Instrument Co., Yellow Spring, Ohio) at 10 [+ or -] 2 and 15 [+ or -] 2[degrees]C for 48 hours. The geckos then were killed and their livers excised, washed in ice-cold 150 mM NaCl (w/v), blotted, weighed and quickly frozen below 30[degrees]C until analyzed. All subsequent procedures were carried out at 4[degrees]C. All chemicals for the assays were of reagent grade and obtained from Sigma Chemical Company, St. Louis, Missouri.
For the determination of NPSH, primarily GSH, the livers were homogenized in ice-cold 0.25 M sucrose using a motor driven Teflon Potter Elvehjem homogenizer. Macromolecules were precipitated by mixing equal volumes of liver homogenate (20 percent, w/v) and trichloroacetic acid (10 percent, w/v) containing 5 mM ethylenediamine tetraacetic acid (EDTA), and centrifuged at 5000g for 15 min. GSH was assayed as a major NPSH (Boyd et al., 1979; Farooqui and Ahmed, 1984) according to the method of Ellman (1959) in which the rate of reduction of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) is proportional to the amount of GSH. The assay medium was 0.1 M potassium phosphate buffer, pH 8.0, containing 5 mM EDTA. To this, 200 [micro]L of tissue extract supernatant was added followed by 5 [micro]mol of DTNB at 20-22[degrees]C. Rate of DTNB reduction was determined spectrophotometrically by recording increased absorbance at 412 nm. Each sample was analyzed at least two times. Also, every daily assay included a standard curve with known amounts of GSH. Statistical analysis was accomplished by Student's t-test at P < 0.05.
RESULTS AND DISCUSSION
Liver weights and the hepatic concentrations of GSH in control geckos and those maintained at manipulated temperatures are shown in Table 1. Data demonstrate existence of GSH as nonprotein sulfhydryl in the livers of geckos. When geckos were placed at 10 and 15[degrees]C, GSH concentrations increased significantly (P > 0.05; 151 and 141 percent, respectively) over those of controls. Mansingh (1985) and Storey et al. (1986) reported an increase in free amino acid pool size (30-45 percent) during cold hardening in freeze tolerant insects. Similarly, Aoyagi et al. (1988) reported that exposure to lower ambient temperatures increased protein turnover in chickens. This protein turnover involved both synthesis and degradation, predominantly the latter. These reports suggest that similar metabolism may have prevailed in geckos in our study where degradation of proteins resulted in significantly lower liver weights and elevated GSH levels. Table 2 presents sex differences in concentrations of hepatic GSH in Mediterranean geckos. It is interesting to note that there was no significant difference (P < 0.05) between the weights of livers of males and females; however, livers of females averaged slightly larger than those of males. Females exhibited significantly higher (P < 0.05) concentrations of GSH than did males. The significance of this is unknown. Perhaps females are more tolerant to cellular oxidative damage than males. Alterations in concentrations of cellular endogenous metabolites including ketone bodies were reported as a result of starvation by green lizards (Pontes et al., 1988). Our studies show similar alterations in protein metabolism in response to changes in ambient temperatures and the resulting fasting. Existence of GSH in geckos indicates that other reptiles also might possess similar metabolic pathways of protein metabolism as those in mammals, and that the enzymes required for GSH metabolism, including GSH peroxidase, GSH reductase, and GSH-S-transferase, might be operating. Braddon et al. (1985) have demonstrated the availability of GSH and GSH cycle enzymes in Black Sea bass.
In conclusion, we have reported the existence of hepatic GSH in Mediterranean geckos, the concentrations of which alter in response to changes in ambient temperature. Thus, the Mediterranean gecko appears to use GSH-related biochemical pathways that deal with toxic effects of xenobiotics. These findings are important because geckos are frequently used as animal models for the study of ecological physiology (Hutchinson and Ritchart, 1989).
TABLE 1. Liver weights and the hepatic glutathione concentrations in the Mediterranean gecko, Hemidactylus turcicus. Each data point is mean [+ or -] SD of six geckos. Treatments Liver weights (mg) GSH ([mu]moles/g) Field control geckos 420 [+ or -] 45 46.3 [+ or -] 5 at ambient temperature (30 [+ or -] 4[degrees]C) Geckos incubated 299 [+ or -] 36* 65.4 [+ or -] 4* at 15 [+ or -] 2[degrees]C Geckos incubated 245 [+ or -] 37* 70.0 [+ or -] 3* at 10 [+ or -] 2[degrees]C * P < 0.05 (Student's t-test). TABLE 2. Comparison of liver weights and hepatic GSH concentrations in male and female geckos at ambient temperature (30[degrees]C). Each data point is mean [+ or -] SD of six geckos. Sex Liver weight (mg) GSH ([mu]moles/g) Males 390 [+ or -] 46.7 38.6 [+ or -] 4.1 Females 421 [+ or -] 48.9 53.7 [+ or -] 3.9* * P < 0.05 (Student's t-test).
This research was partially supported by the University of Texas Pan American Faculty Research Council award to M. Farooqui.
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ROY A. BLOOM AND MOHAMMED Y. H. FAROOQUI
Department of Biology, The University of Texas Pan American, Edinburg, Texas 78539
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|Author:||Bloom, Roy A.; Farooqui, Mohammed Y.H.|
|Publication:||The Texas Journal of Science|
|Date:||Feb 1, 1992|
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