Mechanisms of Ageing and Development 100 (1998) 261 – 267
Glyoxalases activity during Bufo bufo embryo development Fernanda Amicarelli a, Paolo Sacchetta b, Sabrina Colafarina a, Stefania Angelucci b, Michele Miranda a, Carmine Di Ilio b,* a
Dipartimento di Biologia di Base ed Applicata, Uni6ersita` Di L’Aquila, 67100 Coppito, 67100 L’Aquila, Italy b Dipartimento di Scienze Biomediche, Uni6ersita` ‘‘G. D’Annunzio’’, 66100 Chieti, Italy
Received 30 July 1997; received in revised form 9 October 1997; accepted 10 October 1997
Abstract In this work we have investigated the expression of glyoxalase I (GLO I) and glyoxalase II (GLO II) activities during Bufo bufo embryo development and in some tissues of both male and female adult animals, in order to study how they correlate with cell proliferation and differentiation. The results show that both the activities are expressed at significant levels from the earliest developmental stages, reaching the highest values at the end of embryonic development (stage 25). The GLO I/GLO II ratio is very high at the beginning of the development and then gradually decreases as the development goes on. These data emphasize the importance of GLO I activity in the phases in which elevated cell division is taking place. In the differentiated tissues, a peculiar sexual dimorphism in both GLO I and GLO II activities, with higher values in female than in male, was found. GLO I embryonic activity levels are comparable to those found in female differentiated tissues, but significantly higher than those detected in male differentiated tissues. On the contrary, the GLO II activities found in the adult tissues were always higher than those found in embryos. These results further support the idea that high GLO I/GLO II ratios are a characteristic of the proliferative status, which assures a good scavenging action against the potentially cytotoxic and cytostatic effect of methylglyoxal. © 1998 Elsevier Science Ireland Ltd. All rights reserved. * Corresponding author. Tel: +39 871 3555274; fax: +39 871 3555356; e-mail:
[email protected] 0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 0 1 4 8 - 6
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Keywords: Glyoxalase; Bufo bufo; Embryo development; Cell proliferation; Cell differentiation
1. Introduction The glyoxalase system catalyses the conversion of 2-oxoaldehydes, including methylglyoxal, into 2-hydroxycarboxylic acid (Vander Jagt, 1988; Thornalley, 1990a). It comprises two enzymes, glyoxalase I and glyoxalase II, and requires a catalytic amount of GSH (Vander Jagt, 1988; Thornalley, 1990a). Glyoxalase I (GLO I; EC 4.4.1.5) is capable of forming S-(2-hydroxyacyl)glutathione from a number of 2-oxoaldehydes in the presence of GSH, whereas glyoxalase II (GLO II; EC 3.1.2.6) is able to hydrolyse the glutathione thiolesters formed in GLO I reaction to 2-hydroxyacids and glutathione (Vander Jagt, 1988; Thornalley, 1990a). Thus, working in concert the two enzymes convert the electrophilic and cytotoxic 2-oxoaldehydes into less-reactive compounds. In fact, methylglyoxal and S-D-lactoylglutathione inhibit cell growth (Szent-Gyorgyi, 1968), modulate microtubule assembly (Gillespie, 1975) and also play a role in the development of diabetic complications (Thornalley, 1990b) and cancer (Thornalley, 1995). Thus, the glyoxalase enzymes have to be considered as a component of the glutathione-dependent detoxication system capable of protecting cells against the products of oxidative metabolism. Age-related changes in the activity of the glyoxalase system have been described (McLellan and Thornalley, 1989; Sharma-Luthra and Kale, 1994). However, relatively little is known about the level of these enzymes during embryo development and their possible changes occurring in the transition from embryonic to adult life. In the present study the specific activities of GLO I and GLO II were measured in the cytosolic fraction prepared from Bufo bufo embryos during development. For comparison the activity of the glyoxalase system was also measured in some tissues of male and female adult animals, because of the different metabolic peculiarities of the adult tissues compared to embryonic ones, where proliferating cells need to express high glycolytic activity and, as a consequence, may have high methylglyoxal levels.
2. Materials and methods
2.1. Embryos Bufo bufo adults were collected near L’Aquila (Italy); ovulation and fertilization occurred in the laboratory. The eggs were kept at 12–14°C in tap water. Jelly was removed from early embryonic stages by treatment with 0.5% sodium thioglycolate at pH 8.6 in a ratio of 1:1 (v/v). The embryos were staged by reference to Rugh (1962).
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2.2. Cytosol preparation Each cytosol preparation was obtained from the homogenization of 1 ml of embryos (from 50 to 130 embryos according to the stage). The values of each time point investigated represent the mean of four separate preparations. The embryos, after three washes with 0.1 M sodium phosphate buffer at pH 7.0, were suspended in the same medium up to 5 ml and homogenized with Potter Elvehjem homogenizer by hand with 10 pestle strokes, except for stage 25 when the pestle was motor driven. The homogenate was centrifuged at 4°C for 60 min at 105 000× g, and the supernatant recovered by syringe to avoid contamination by the upper lipid layer and used for enzymatic measurements. Tissues were suspended in 10 vol of 0.1 M potassium phosphate buffer, pH 7.0, and homogenized with a Potter Elvehjem homogenizer by hand with 10 pestle strokes. Cytosols were prepared by centrifugation at 105 000× g for 60 min at 4°C.
2.3. Assays GLO I activity was monitored as described by Mannervik et al. (1988). The assay solution contained 0.1 mM potassium phosphate buffer (pH 7.0), 2 mM methylglyoxal, 1 mM GSH and appropriate amounts of enzyme solution. Enzyme activity was monitored at 25°C following the increase in absorbance at 240 nm, and expressed as nmol of S-D-lactoylglutathione produced/min per mg of protein. GLO II activity was assayed by measuring the initial rate of hydrolysis of S-D-lactoylglutathione to GSH and D-lactic acid. The assay solution contained 0.5 mM Tris/HCl buffer (pH 7.5), 300 mM of S-D-lactoylglutathione and appropriate amounts of enzyme solution. Enzyme activity was monitored at 25°C following the decrease in absorbance at 240 nm, and expressed as nmol/min per mg of protein. Protein concentration was determined by the method of Bradford (1976) using g-globulin as standard.
3. Results and discussion The results of a typical experiment showing GLO I and GLO II activity profiles during the development of Bufo bufo embryos are reported in Tables 1–3. The data obtained demonstrate that both activities are present in a significant amount from the early stages of development. It is interesting to note that the level of GLO I and GLO II activities found in amphibian embryos are comparable to those measured in day 10 cultured mouse embryos (Tiboni et al., unpublished data). In addition to the enzymes of the glyoxalase system, other GSH- and non-GSH-related enzymes of the antioxidant system, including glutathione peroxidase (Di Ilio et al., 1984), glutathione reductase (Di Ilio et al., 1984), glucose 6-phosphate dehydrogenase (Miranda, 1976), glutathione transferase (Del Boccio et al., 1987; Miranda et al., 1987; Aceto et al., 1993), and catalase (Miranda et al., 1987), are early expressed in Bufo bufo embryos. Thus, amphibian embryos are well equipped to defend them-
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Table 1 Glyoxalase I and glyoxalase II activities during Bufo bufo embryo development Stage
3 – 4 (130) 7 (130) 9 (130) 12 (110) 14 (80) 18 (110) 20 (80) 23 (50) 25 (50)
GLO I
GLO II
GLO I/GLO II ratio
mU/embryo
mU/mg
mU/embryo
mU/mg
409 2.0 3191.0* 259 2.0* 309 1.0* 239 2.0* 219 0.2* 239 2.0* 2692.0* 5491.0*
22499.0 2009 6.0 14399.0 21193.0 19790.0 19790.0 9696.0 1559 6.0 3119 6.0
2.17 90.14 1.10 9 0.02* 0.73 90.03* 1.26 90.08* 1.10 90.00* 3.30 90.22* 3.10 90.30* 3.70 9 0.20* 8.20 9 0.40**
12 9 0.8 7.0 9 0.2 4.0 9 0.2 8.7 90.6 8.10 9 0.0 81 9 0.0 13.1 91.0 22 9 1.0 48 92.0
18.43 28.18 34.25 23.80 20.90 6.36 7.41 7.03 6.58
Values reported are means 9S.E.M of four different determinations. The values reported in parentheses indicate the number of embryos used for each determination. *pB0.05 with respect to the activity of stages 3 – 4 using the paired Student’s t-test; **pB0.025 with respect to the activity of stages 3–4 using the paired Student’s t-test.
selves, during all phases of development, against the deleterious effects of toxic metabolites arising from oxidative metabolism. Studying the profile of GLO I activity (as expressed per nmol/min/embryo), we could describe approximately two periods. In fact, a gradual decrease of GLO I activity, from stage 3 to stage 14, followed by a slight increase up to the end of development can be noted. The maximum level of GLO I activity was obtained at stage 25. In contrast to GLO I, more pronounced fluctuations of GLO II activity seem to occur during Bufo bufo embryo development. Three periods could be approximately described as: a slight fall from stage 3 to stage 9, when a minimum is reached; a gradual increase from stage 9 to stage 23; and a sharp rise from stage 23 to stage 25. As for GLO I, the maximum level of GLO II activity was found at stage 25. The relatively higher amount of both GLO I and GLO II activities present at stage 25 may be ascribed to the achievment of terrestrial life and to changes in feeding that, from an endogenous source, becomes dependent on external sources. The ratio of the Table 2 Glyoxalase I and glyoxalase II activities in tissues of male Bufo bufo adults Tissue
GLO I (mU/mg)
GLO II (mU/mg)
GLO I/GLO II ratio
Liver Lung Muscle Heart
69.759 2.85* (n= 5) 110.495.3 (n= 5) 85.490.2* (n=4) 59.9 92.9* (n= 6)
53.159 1.8 (n = 5) 13.6 90.6* (n = 5) 17.4 9 1.3** (n = 4) 15.6 9 0.2* (n =6)
1.28 8.12 4.90 3.84
Values reported are means 9S.E.M. n= number of samples investigated. *pB0.01 vs. female, using the paired Student’s t-test; **pB0.05 vs. female, using the paired Student’s t-test.
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Table 3 Glyoxalase I and glyoxalase II activities in tissue of female Bufo bufo adults Tissue
GLO I (mU/mg)
GLO II (mU/mg)
GLO I/GLO II ratio
Liver Lung Muscle Heart Ovary
201.2 9 11* (n= 5) 89.39 4.2 (n= 5) 254.8 9 15.5* (n = 4) 259.9 9 3.8* (n= 6) 193.39 8.7 (n= 6)
58.45 9 3.1 (n =5) 22.8 9 0.70* (n = 5) 22.1 90.4** (n = 4) 40.2 9 0.9* (n = 6) 47.7 90.7 (n = 6)
3.53 3.92 11.52 6.46 4.05
Values reported are means 9 S.E.M. n =number of samples investigated. *pB0.01 vs. male, using the paired Student’s t-test; **pB0.05 vs. male, using the paired Student’s t-test.
activities of GLO I and GLO II (GLO I/GLO II) is higher at the beginning than at the end of development, suggesting the importance of GLO I activity in the phases in which a rapid rate of cell division is taking place, with concomitant expansion of new cellular clones that need a high control of cell proliferation and an efficient detoxication process. In this connection, it is worth noting that increased levels of GLO I activities were also found during liver regeneration following partial hepatectomy in both rats and mice (Dixit et al., 1983; Principato et al., 1983). Accordingly, in chickens, high GLO I activity in immature proliferating tissues and low activity in mature differentiated tissues was found (Principato et al., 1982). Furthermore, when compared to non-malignant quiescent cells, several tumor cell lines also display high levels of GLO I activity (Jerzykowski et al., 1978). For comparison, the glyoxalase system has also been measured in some tissues of both male and female Bufo bufo adults. Sex-associated changes in the expression of the enzymes of the glyoxalase system were found. In fact, analysis of the results obtained indicate that both GLO I and GLO II activities are higher in female tissues than in the corresponding adult male tissues. The only exception was found in the lung, where the level of GLO I activity in females is essentially similar to that found in males. The reasons for this difference are unknown at present. However, it is possible that the expression of the enzymes of glyoxalase system may be, at least in part, regulated by sexual factors. In the liver of Rana esculenta three different GLO II isoforms with isoelectric points at pH 8.70, 8.40 and 8.20 were found (Principato et al., 1987). The possibility that the single tissue of adult Bufo bufo possesses a different amount of each of the three GLO II isoforms could not be ruled out. At present we are in the process of verifying this hypothesis. In this connection, it is worth noting that, in several amphibian tissues, a different expression of the isoforms of glutathione transferase was also found (Aceto et al., 1993). In male Bufo bufo adult, the lung is the tissue showing highest GLO I activity, followed by muscle, heart and liver. On the contrary, in females, the heart is the tissue showing highest GLO I activity, followed by muscle, liver, ovary and lung. In comparison with the activity of several other organs, a relatively low level of GLO I activity was also found in human female liver (Jerzykowski et al., 1978; Larsen et al., 1985). For both, male and female Bufo bufo adults, the liver is the tissue showing the highest level of GLO II activity. A high level of GLO II activity
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was also found in the liver of different vertebrate species including man (Jerzykowski et al., 1978). It has to be noted that the activity values of GLO I found in the embryos of Bufo bufo during development are higher than those found in the male adult tissues, but comparable with those present in female adult tissues. On the contrary, higher levels of GLO II activity were found in the adult tissues, as compared with those found in the embryos. Thus, these results further confirm the notion that a high level of GLO I activity, and a low level of GLO II activity, are characteristics of the proliferative status and that the reverse occurs in the differentiated state. In conclusion, a gradual increase of both GLO I and GLO II activity was seen during Bufo bufo embryo development. Furthermore, the transition from the embryonic to adult life induces an increase of GLO II activity with a concomitant decrease of GLO I activity in males but not in females.
Acknowledgements This work in part supported by the CNR grant n. 96.03351.CT04
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