J. Biochem., 79, 153-158 (1976)
Effects of Phthalate Esters on the Respiration of Rat Liver Mitochondria Tohru OHYAMA Hokkaido Institute of Public Health, N 19, W 12, Kita-ku, Sapporo, Hokkaido 060 Received for publication, May 12, 1975
The effects of phthalate esters on the oxidation of succinate, glutamate, /3-hydroxy"butyrate and NADH by rat liver mitochondria were examined and it was found that •di-w-butyl phthalate (DBP) strongly inhibited the succinate oxidation by intact and sonicated rat mitochondria, but did not inhibit the State 4 respiration with NADlinked substrates such as glutamate and /3-hydroxybutyrate. However oxygen uptake accelerated by the presence of ADP and substrate (State 3) was inhibited and the rate of oxygen uptake decreased to that without ADP (State 4). It was concluded that phthalate esters were electron and energy transport inhibitors but not uncouplers. Phthalate esters also inhibited NADH oxidation by sonicated mitochondria. The degree of inhibition depended on the carbon number of the alkyl groups of phthalate esters, and DBP was the most potent inhibitor of respiration. The activity of purified beef liver glutamate dehydrogenase [EC 1.4.1.3] was slightly inhibited by phthalate esters.
Environmental pollution by phthalate esters has become worse with the increasing production of plastics. Phthalate esters have been detected not only in water, soil, air, and foods, but also in animal organs. To estimate the toxicity of phthalate esters, many studies of its distribution, metabolism, excretion, and accumulation in rats ( 7 ) and aquatic invertebrates (2) have been carried out. However phthalate esters have been shown to be of low toxicity (3, 4). Nazir et al. ( 5 ) reported the Abbreviations: DMP, dimethyl phthalate; DEP, diethyl phthalate ; DPP, di-n-propyl phthalate ; DBP, di-n-butyl phthalate; DIBP, diisobutyl phthalate; DHP, dihexyl phthalate; DOP, dioctyl phthalate; DEHP, di-2-ethylhexyl phthalate. Vol. 79, No. 1, 1976
153
specific localization of di-2-ethylhexyl phthalate in bovine heart muscle mitochondria and their studies on intracellular distribution have shown that phthalate esters may act on the mitochondrial membrane, particularly on the lipid part. Dillingham et al. (6) have reported that in toxicity at the intercellular level, destabilization of the membrane is the most important effect of phthalate esters. The acute and subacute toxicity of phthalate esters has been studied in detail in recent years from physiological, genetic, and morphological viewpoints (7—5), but fewer studies have been made from the viewpoint of molecular biology. In order to understand the toxicological implications of phthalate esters in more detail,
154
T. OHYAMA.
we shall consider the effect of phthalate esters on the respiration of rat liver mitochondria in this paper. MATERIALS AND METHODS Rat liver mitochondria were prepared by the method of Schneider (10). Male rats of the Wistar strain, weighing 300—400 g, were used and livers were disrupted manually in 0.25 M sucrose and 10 mM Tris-HCl (pH 7.3) with a Potter-Elvehjem homogenizer. Sonicated mitochondria were prepared by the sonic disruption of intact mitochondria at 0° for 5 min at 10 kHz using a Kubota KMS-100 sonicater. Intact and sonicated mitochondria were suspended in an ice-cold solution containing 0.25 M sucrose and 10 mM Tris-HCl (pH 7.3) to give a concentration of 20 mg of protein/ml. Protein concentration was determined by the method of Lowry et al. (11) using bovine serum albumin as a standard. Respiration rates were measured at 25° with a Clark-type oxygen electrode purchased from Beckmann. The reaction vessel with an oxygen electrode was modified from that of Estabrook (12) and was made in the Research Institute of Applied Electricity, Hokkaido University ; the vessel volume was 2.25 ml. The reaction mixture contained 0.25 M sucrose, 10 mM Tris-HCl (pH 7.3), 5 mM potassium phosphate buffer (pH 7.3), 10 mM KC1, 5 mM MgClj, and 0.2 mM EDTA. Oxygen uptake was recorded after the addition of 0.05 ml of mitochondrial suspension (1 mg of mitochodrial protein) then 11.1 mM succinate or glutamate, 0.56 mM /3-hydroxybutyrate and 2.2 mM NADH in succesion. Other experimental details are given in the figures and table. Phthalate esters used in all experiments were dissolved in ethanol; dissolved molecular oxygen was excluded by bubbling nitrogen gas. The rate of NADH oxidation was determined by measuring the decrease of absorbance at 340 nm in a quartz cuvette using a Shimadzu MPS-50L recording spectrophotometer at 25°. The mixture (final volume 5 ml), containing 0.1 M potassium phosphate buffer (pH 7.3), 0.1 mg of sonicated mitochondria, and
various concentrations of phthalate esters, was incubated for 90 min at 25°. The reaction was started by the addition of 0.1 mM NADH to 3 ml of the incubated solution. The same volume of ethanol was added in a control experiment without phthalate esters. Glutamate dehydrogenase [EC 1.4.1.3] activity was measured at 25° by following the absorbance at 340 nm. The mixture (final volume 3.0 ml), containing 0.1 M potassium phosphate buffer (pH 7.3), 20 fig of the enzyme, and various concentrations of phthalate esters, was incubated for 90 min and the reaction was started by the addition of 0.1 mM NAD+ and 50 mM glutamate to the solution. Bovine liver glutamate dehydrogenase was obtained from Boehringer Mannheim as a glycerol solution. The concentration of the enzyme in the medium was calculated from the absorbance according to Olson and Andinsen (13). All commercial reagents used were of analytical grade. The purity of phthalate esters was checked by gas chromatography and thin layer chromatography. Dihexyl phthalate was synthesized by Dr. Ogawa in this institute from commercial phthalic acid and hexyl alcohol of analytical grade. RESULTS Effects of Phthalate Esters on the Oxygen Uptake by Intact Mitochondria with Succinate as a Substrate—The respiratory control index of intact mitochondria used in this experiment was approximately 3.5. The effects of phthalate esters on respiration in the presence of ADP and substrate (State 3) was examined. The oxygen uptake was inhibited to less than half of the control value by the presence of 222 ppm of each phthalate ester. As shown in Fig. 1, the effect of DBP was greatest. Next was DHP, followed by DPP, DOP, DMP, and DEP. Figure 2 shows the relation between Jo inhibition in State 3 and State 4 and the carbon number of the alkyl groups of phthalate esters. It was noticeable that DBP, containing 4 carbon atoms in the phthalate ester alkyl group was the most potent inhibitor of oxygen uptake in both State 3 and / . Biochem.
EFFECTS OF PHTHALATES ON MITOCHONDRIA
Sue
155
•ADP
100/jMOt
1 DBP -5 mm
Fig. 1. Effect of phthalate esters on the State 3 respiration rate of intact mitochondria in the presence of succinate. Mitochondria, succinate, ADP, and phthalate esters were added at the points indicated by arrows. Final concentrations of succinate, ADP, and phthalate esters were 11.1 mM, 0.7 mM, and 222 ppm, respectively (222 ppm of DBP is 0.8 mM).
, 2 1O"3M
Fig. 3. Effect of the concentration of DBP on the respiration rates in States 3 and 4 in the presence of succinate. O : % inhibition in State 3 respiration with succinate and ADP. • : % inhibition in State 4 respiration with succinate alone as a substrate. The rate of inhibition was measured from the traces shown in Fig. 1. Final concentrations of succinate and ADP were 11.1 mM and 0.7 mM, respectively. Mito. Glu.
ADP
1 2 3 A 5 6 7 Carbon Number of Alkyl Group
8
Fig. 2. Relation between % inhibition in States 3 and 4 and the carbon number of the alkyl groups of phthalate esters. Final concentrations of succinate and ADP were 11.1 mM and 0.7 mM, respectively. O : % inhibition in State 3 respiration calculated from the data shown in Fig. 1. • : % inhibition in State 4 respiration. The final concentration of phthalate esters was 0.8 mM.
State 4. The relationship between the inhibition and the concentration of DBP in State 3 and State 4 is shown in Fig. 3. In the presence of approximately 3 mM DBP, the inhibition of State 3 respiration was nearly complete; the DBP concentration giving 50% inhibition was 0.32 mM. In the case of State 4 respiration, the inhibition reached about 80% in the presence of 1.2 mM DBP and the concentration giving 50% inhibition was 0.64 mM. Effects of Phthalate F'ler* on the OxidaVol. 79, No. 1, 1976
5min
Fig. 4. Effect of DBP on glutamate oxidation by intact mitochondria. Final concentrations of glutamate, ADP, and DBP were 11.1 mM, 0.7 mM, and 0.8 mM, respectively. Mitochondria, glutamate, ADP, and DBP were added at the points indicated by arrows.
tion of NAD-linked Substrates—The respiratory control indices of the mitochondria used with glutamate and /3-hydroxybutyrate were approximately 2.0 and 2.4, respectively. Figure 4a shows that, unlike the case of succinate oxidation, 0.8 mM DBP did not affect the oxygen uptake in the presence of glutamate alone. In Fig. 4b, the effect of DBP on the oxygen uptake in the presence of glutamate and succinate is shown. The addition of DBP selectively suppressed the oxygen uptake caused by succinate. The effect of DBP on State 3 respiration with glutamate is shown in Fig. 4c.
156
T. OHYAMA Mito. ' 0-OHButyrate DBP 50>iM-0: Sue.
5 min
—*
Fig. 5. Effect of DBP on 0-hydroxybutyrate oxidation as an NAD-linked substrate. Final concentrations of /9-hydroxybutyrate, ADP, and DBP were 11.1 mM, 0.7 mM, and 0.8 mM, respectively. Mitochondria, /5-hydroxybutyrate, ADP, and DBP were added at the points indicated by arrows.
2 3 A 5 6 7 Carbon Number of Alkyl Group
time.min
Fig. 7. Effect of DBP on NADH oxidation by sonicated mitochondria. Sonicated mitochondria, NADH, succinate, DBP, and KCN were added at the points indicated by arrows and their final concentrations were 2mg of protein, 2.2 mM, 11.1 mM, 0.8 mM, and 4.4 mM, respectively.
8
Fig. 6. Relation between % inhibition of succinate oxidation by sonicated mitochondria and the carbon number of the alkyl groups of phthalate esters. Final concentrations of succinate and phthalate esters were 11.1 mM and 0.8 mM, respectively.
The addition of DBP eliminated the accelerated oxygen uptake in the presence of ADP and the rate of oxygen uptake decreased to that without ADP. Therefore DBP appeared to inhibit the oxygen uptake only in State 3 in the case of glutamate. Figure 5 shows the effect of DBP on the oxygen uptake by intact mitochondria in the presence of ^-hydroxybutyrate as a NAD-linked substrate. DBP inhibited the oxidation of /S-hydroxybutyrate only in State 3, as indicated in Fig. 5a. DBP did not exhibit an inhibitory effect on respiration in the presence of the substrate only, just as in the case of glutamate oxidation. Effects of Phthalate Esters on the Oxygen Uptake by Sonicated Mitochondria in the Presence of Succinate or NADH— Phthalate esters inhibited the oxidation of succinate, and the
50
100
150
( min )
Fig. 8. Time-dependent inhibition by DOP of the NADH oxidase activity of sonicated mitochondria. Aliquots of the incubated mixture were withdrawn at the indicated times and the activity was measured spectrophotometrically. The final concentration of DOP was 0.128 mM. See "MATERIALS AND METHODS."
inhibition pattern depended on the carbon number of the alkyl groups, as observed with intact mitochondria. A difference of inhibition between straight and branched chain alkyl groups was present, as shown in Fig. 6. The dependence of succinate oxidation by sonicated mitochondria on the concentration of DBP, which was the most potent inhibitor, was also examined. It was found that the inhibition by 3 mM DBP was nearly complete, as in the case of State 3 oxygen uptake by intact mitochondria, and the concentration giving 50% inhibition was 0.18 mM. Figure 7 shows the effect of phthalate es/. Biochem.
EFFECTS OF PHTHALATES ON MITOCHONDRIA
ters on the oxidation of NADH by sonicated mitochondria. DBP inhibited the oxidation of succinate and NADH. The dependence of the inhibition on the carbon number of the alkyl groups was similar to that shown in Fig. 6. The effects of phthalate esters on the NADH oxidase activity of sonicated mitochondria were examined spectrophotometrically. In this case, the degree of inhibition depended on incubation time, probably because a lower concen-
157
tration of sonicated mitochondria was used than in the activity assay by the oxygen electrode method. From Fig. 8, it was found that more than 90 min was necessary for the completion of inhibition when DOP was used. The concentrations of phthalate esters at which NADH oxidase activity fell to 50% were determined. The relation between the logarithm of the concentrations of phthalate esters giving 50% inhibition and the carbon numbers of the alkyl groups is shown in Fig. 9. These results are analogous to those obtained by the oxygen electrode method. Effects of Phthalate Esters on Purified Beef Liver Glutamate Dehydrogenase—Table I shows the effects of different phthalate esters on the activity of beef liver glutamate dehydrogenase. Under the conditions employed, only slight inhibition was observed with DEP, DBP, and DIBP. DISCUSSION
1 2 3 A 5 6 7 8 Carbon Number of Alkyl Group ot Phthalate Esters
Fig. 9. Plot of the logarithm of inhibition constants of phthalate esters in NADH oxidation by sonicated mitochondria against the carbon number of their alkyl groups. The inhibition constants were obtained as the concentrations of phthalate esters giving 50% inhibition after incubation for 90 min (Fig. 8). TABLE I. Effects of phthalate esters on purified beef liver glutamate dehydrogenase. Experimental conditions are given in " MATERIALS AND METHODS." 100 ppm phthalate
Activity
% Inhibition
Dimethyl Diethyl
0.588 0.522
13.0
Di-«-butyl Dihexyl Dioctyl Diisobutyl Diethylhexyl Control
0.504 0.576 0.570 0.540 0.570 0.600
Vol. 79, No. 1, 1976
2.0
16.0 4.0 5.0
10.0 5.0 —
It is considered that there are two kinds of inhibition by phthalate esters of oxygen uptake by intact mitochondria. One occurs at the succinate oxidation site of the electron transport system. This is shown by the fact that the oxygen uptake in State 4 was inhibited when the substrate was succinate but was not inhibited when it was glutamate or /9-hydroxybutyrate. The other is the inhibition of energy transport which is associated with State 3. This is shown by the fact that the oxygen uptake in State 3 was strongly inhibited by phthalate esters in the presence of either succinate or an NAD-linked substrate, and the rate of oxygen uptake decreased to the level of State 4. However, it still remains to be determined which site phthalate esters act on. In the case of sonicated mitochondria, NADH oxidation as well as succinate oxidation was inhibited by phthalate esters. It might be said that the NADH oxidation site can interact with phthalate esters because of the destruction of the mitochondrial membrane by sonication. The biological effect of the carbon number of the alkyl groups of phthalate esters was investigated by Sugawara (14). In his
158
report, five phthalate esters were tested for effect on the hatching of brine shrimp eggs and the order of toxicity was DBP>DHP> DEP>DOP>DMP. Moreover, Kazuya (75) demonstrated that the order of toxicity of phthalate esters was DBP>DEP>DMP using fibroblast cultures from newborn rat cerebellar tissure. However, it was not determined which chemical or physical feature of the phthalate esters was responsible for their toxicity. The results reported here suggest that the toxicity of phthalate esters cannot be determined by their water solubility (16) alone. Calley et al. ( 77), studying the effect of administration i.v. of three phthalate esters (DMP, DEP, DIBP) on the respiratory rate of the anesthetized rabbit, reported that there was a significant increase in respiratory rate after the administration of a total dose of 100 mg/kg and also that the rate gradually returned to the normal level over a period of about 5 min. This is inconsistent with our result that 0.5—2.0 mM DBP, corresponding to 139-556 mg/kg, strongly inhibited the respiration of rat liver mitochondria. The apparent contradiction to be resolved. The potent inhibitory effect of DBP might be explained in terms of specific interaction between the alkyl group of 4 carbon atoms and the inhibitory site. However the details of the mechanism require further experiments for their elucidation. The author's thanks are due to Prof. Dr. I. Yamazaki, Research Institute of Applied Electricity, Hokkaido University for advice, and to Drs. H. Kanashima and Y. Kinoshita, Hokkaido Institute of Public Health, for their help.
T. OHYAMA
REFERENCES 1. Schultz, CO. & Rubin, R.J. (1973) Environmental Perspective 123-129 2. Mayer, Jun. F.L., Stalling, D.L., & Johnson, J.L. (1972) Nature 238, 411-413 3. Hodge, H.C. (1943) Proc. Soc. Exp. Biol. Med. 53, 20-23 4. Harris, R.S., Hodge, H.C, Maynard, E.A., & Blanchet, H.J. (1956) AM A Arch. Indust. Health 13, 259-264 5. Nazir, D.J., Alcraz, A.P., Bierl, B.A., Beroza, M., & Nair, P.P. (1971) Biochemistry 10, 42284232 6. Dillingham, E.O., WuCheng-Hsien, & Autian, J. (1972) Toxicol. Appl. Pharmacol. 22, 318 7. Snigh, A.R., Lawrence, W.H., & Autian, J. (1972) / . Pharm. Sri. 61, 51-55 8. Patty, E.A. (1969) Industrial Hygiene & Toxicology II, 1903 9. Smith, C.C. (1953) AM A Arch. Indust. Hyg. & Occup. Med. 7, 310 10. Schneider, W.C (1948) / . Biol. Chem. 176, 256 11. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) /. Biol. Chem. 193, 265-275 12. Estabrook, R.W. (1967) in Methods in Enzymology (Estabrook, R.W. & Pullman, M.E., eds.) Vol. X, pp. 41-47, Academic Press, New York 13. Olson, J.A. & Andinsen, C.B. (1952) / . Bid. Chem. 197, 67-89 14. Sugawara, N. (1974) Toxicol. Appl. Pharmacol. 30, 87-89 15. Kazuya, M. (1973) Cytotoxirity of phthalate esters, Jap. J. Hyg. 28, 248-252 16. Fishbein, L. & Albro, D.W. (1972) / . Chromatography 70, 365-412 17. Calley, D., Autian, J., & Wallace, G.W. (1966) / . Pharm. Sri. 55, 158-162
/ . Biochem.
Effects of Phthalate Esters on the Respiration of Rat Liver Mitochondria Tohru OHYAMA Hokkaido Institute of Public Health, N 19, W 12, Kita-ku, Sapporo, Hokkaido 060 Received for publication, May 12, 1975
The effects of phthalate esters on the oxidation of succinate, glutamate, /3-hydroxy"butyrate and NADH by rat liver mitochondria were examined and it was found that •di-w-butyl phthalate (DBP) strongly inhibited the succinate oxidation by intact and sonicated rat mitochondria, but did not inhibit the State 4 respiration with NADlinked substrates such as glutamate and /3-hydroxybutyrate. However oxygen uptake accelerated by the presence of ADP and substrate (State 3) was inhibited and the rate of oxygen uptake decreased to that without ADP (State 4). It was concluded that phthalate esters were electron and energy transport inhibitors but not uncouplers. Phthalate esters also inhibited NADH oxidation by sonicated mitochondria. The degree of inhibition depended on the carbon number of the alkyl groups of phthalate esters, and DBP was the most potent inhibitor of respiration. The activity of purified beef liver glutamate dehydrogenase [EC 1.4.1.3] was slightly inhibited by phthalate esters.
Environmental pollution by phthalate esters has become worse with the increasing production of plastics. Phthalate esters have been detected not only in water, soil, air, and foods, but also in animal organs. To estimate the toxicity of phthalate esters, many studies of its distribution, metabolism, excretion, and accumulation in rats ( 7 ) and aquatic invertebrates (2) have been carried out. However phthalate esters have been shown to be of low toxicity (3, 4). Nazir et al. ( 5 ) reported the Abbreviations: DMP, dimethyl phthalate; DEP, diethyl phthalate ; DPP, di-n-propyl phthalate ; DBP, di-n-butyl phthalate; DIBP, diisobutyl phthalate; DHP, dihexyl phthalate; DOP, dioctyl phthalate; DEHP, di-2-ethylhexyl phthalate. Vol. 79, No. 1, 1976
153
specific localization of di-2-ethylhexyl phthalate in bovine heart muscle mitochondria and their studies on intracellular distribution have shown that phthalate esters may act on the mitochondrial membrane, particularly on the lipid part. Dillingham et al. (6) have reported that in toxicity at the intercellular level, destabilization of the membrane is the most important effect of phthalate esters. The acute and subacute toxicity of phthalate esters has been studied in detail in recent years from physiological, genetic, and morphological viewpoints (7—5), but fewer studies have been made from the viewpoint of molecular biology. In order to understand the toxicological implications of phthalate esters in more detail,
154
T. OHYAMA.
we shall consider the effect of phthalate esters on the respiration of rat liver mitochondria in this paper. MATERIALS AND METHODS Rat liver mitochondria were prepared by the method of Schneider (10). Male rats of the Wistar strain, weighing 300—400 g, were used and livers were disrupted manually in 0.25 M sucrose and 10 mM Tris-HCl (pH 7.3) with a Potter-Elvehjem homogenizer. Sonicated mitochondria were prepared by the sonic disruption of intact mitochondria at 0° for 5 min at 10 kHz using a Kubota KMS-100 sonicater. Intact and sonicated mitochondria were suspended in an ice-cold solution containing 0.25 M sucrose and 10 mM Tris-HCl (pH 7.3) to give a concentration of 20 mg of protein/ml. Protein concentration was determined by the method of Lowry et al. (11) using bovine serum albumin as a standard. Respiration rates were measured at 25° with a Clark-type oxygen electrode purchased from Beckmann. The reaction vessel with an oxygen electrode was modified from that of Estabrook (12) and was made in the Research Institute of Applied Electricity, Hokkaido University ; the vessel volume was 2.25 ml. The reaction mixture contained 0.25 M sucrose, 10 mM Tris-HCl (pH 7.3), 5 mM potassium phosphate buffer (pH 7.3), 10 mM KC1, 5 mM MgClj, and 0.2 mM EDTA. Oxygen uptake was recorded after the addition of 0.05 ml of mitochondrial suspension (1 mg of mitochodrial protein) then 11.1 mM succinate or glutamate, 0.56 mM /3-hydroxybutyrate and 2.2 mM NADH in succesion. Other experimental details are given in the figures and table. Phthalate esters used in all experiments were dissolved in ethanol; dissolved molecular oxygen was excluded by bubbling nitrogen gas. The rate of NADH oxidation was determined by measuring the decrease of absorbance at 340 nm in a quartz cuvette using a Shimadzu MPS-50L recording spectrophotometer at 25°. The mixture (final volume 5 ml), containing 0.1 M potassium phosphate buffer (pH 7.3), 0.1 mg of sonicated mitochondria, and
various concentrations of phthalate esters, was incubated for 90 min at 25°. The reaction was started by the addition of 0.1 mM NADH to 3 ml of the incubated solution. The same volume of ethanol was added in a control experiment without phthalate esters. Glutamate dehydrogenase [EC 1.4.1.3] activity was measured at 25° by following the absorbance at 340 nm. The mixture (final volume 3.0 ml), containing 0.1 M potassium phosphate buffer (pH 7.3), 20 fig of the enzyme, and various concentrations of phthalate esters, was incubated for 90 min and the reaction was started by the addition of 0.1 mM NAD+ and 50 mM glutamate to the solution. Bovine liver glutamate dehydrogenase was obtained from Boehringer Mannheim as a glycerol solution. The concentration of the enzyme in the medium was calculated from the absorbance according to Olson and Andinsen (13). All commercial reagents used were of analytical grade. The purity of phthalate esters was checked by gas chromatography and thin layer chromatography. Dihexyl phthalate was synthesized by Dr. Ogawa in this institute from commercial phthalic acid and hexyl alcohol of analytical grade. RESULTS Effects of Phthalate Esters on the Oxygen Uptake by Intact Mitochondria with Succinate as a Substrate—The respiratory control index of intact mitochondria used in this experiment was approximately 3.5. The effects of phthalate esters on respiration in the presence of ADP and substrate (State 3) was examined. The oxygen uptake was inhibited to less than half of the control value by the presence of 222 ppm of each phthalate ester. As shown in Fig. 1, the effect of DBP was greatest. Next was DHP, followed by DPP, DOP, DMP, and DEP. Figure 2 shows the relation between Jo inhibition in State 3 and State 4 and the carbon number of the alkyl groups of phthalate esters. It was noticeable that DBP, containing 4 carbon atoms in the phthalate ester alkyl group was the most potent inhibitor of oxygen uptake in both State 3 and / . Biochem.
EFFECTS OF PHTHALATES ON MITOCHONDRIA
Sue
155
•ADP
100/jMOt
1 DBP -5 mm
Fig. 1. Effect of phthalate esters on the State 3 respiration rate of intact mitochondria in the presence of succinate. Mitochondria, succinate, ADP, and phthalate esters were added at the points indicated by arrows. Final concentrations of succinate, ADP, and phthalate esters were 11.1 mM, 0.7 mM, and 222 ppm, respectively (222 ppm of DBP is 0.8 mM).
, 2 1O"3M
Fig. 3. Effect of the concentration of DBP on the respiration rates in States 3 and 4 in the presence of succinate. O : % inhibition in State 3 respiration with succinate and ADP. • : % inhibition in State 4 respiration with succinate alone as a substrate. The rate of inhibition was measured from the traces shown in Fig. 1. Final concentrations of succinate and ADP were 11.1 mM and 0.7 mM, respectively. Mito. Glu.
ADP
1 2 3 A 5 6 7 Carbon Number of Alkyl Group
8
Fig. 2. Relation between % inhibition in States 3 and 4 and the carbon number of the alkyl groups of phthalate esters. Final concentrations of succinate and ADP were 11.1 mM and 0.7 mM, respectively. O : % inhibition in State 3 respiration calculated from the data shown in Fig. 1. • : % inhibition in State 4 respiration. The final concentration of phthalate esters was 0.8 mM.
State 4. The relationship between the inhibition and the concentration of DBP in State 3 and State 4 is shown in Fig. 3. In the presence of approximately 3 mM DBP, the inhibition of State 3 respiration was nearly complete; the DBP concentration giving 50% inhibition was 0.32 mM. In the case of State 4 respiration, the inhibition reached about 80% in the presence of 1.2 mM DBP and the concentration giving 50% inhibition was 0.64 mM. Effects of Phthalate F'ler* on the OxidaVol. 79, No. 1, 1976
5min
Fig. 4. Effect of DBP on glutamate oxidation by intact mitochondria. Final concentrations of glutamate, ADP, and DBP were 11.1 mM, 0.7 mM, and 0.8 mM, respectively. Mitochondria, glutamate, ADP, and DBP were added at the points indicated by arrows.
tion of NAD-linked Substrates—The respiratory control indices of the mitochondria used with glutamate and /3-hydroxybutyrate were approximately 2.0 and 2.4, respectively. Figure 4a shows that, unlike the case of succinate oxidation, 0.8 mM DBP did not affect the oxygen uptake in the presence of glutamate alone. In Fig. 4b, the effect of DBP on the oxygen uptake in the presence of glutamate and succinate is shown. The addition of DBP selectively suppressed the oxygen uptake caused by succinate. The effect of DBP on State 3 respiration with glutamate is shown in Fig. 4c.
156
T. OHYAMA Mito. ' 0-OHButyrate DBP 50>iM-0: Sue.
5 min
—*
Fig. 5. Effect of DBP on 0-hydroxybutyrate oxidation as an NAD-linked substrate. Final concentrations of /9-hydroxybutyrate, ADP, and DBP were 11.1 mM, 0.7 mM, and 0.8 mM, respectively. Mitochondria, /5-hydroxybutyrate, ADP, and DBP were added at the points indicated by arrows.
2 3 A 5 6 7 Carbon Number of Alkyl Group
time.min
Fig. 7. Effect of DBP on NADH oxidation by sonicated mitochondria. Sonicated mitochondria, NADH, succinate, DBP, and KCN were added at the points indicated by arrows and their final concentrations were 2mg of protein, 2.2 mM, 11.1 mM, 0.8 mM, and 4.4 mM, respectively.
8
Fig. 6. Relation between % inhibition of succinate oxidation by sonicated mitochondria and the carbon number of the alkyl groups of phthalate esters. Final concentrations of succinate and phthalate esters were 11.1 mM and 0.8 mM, respectively.
The addition of DBP eliminated the accelerated oxygen uptake in the presence of ADP and the rate of oxygen uptake decreased to that without ADP. Therefore DBP appeared to inhibit the oxygen uptake only in State 3 in the case of glutamate. Figure 5 shows the effect of DBP on the oxygen uptake by intact mitochondria in the presence of ^-hydroxybutyrate as a NAD-linked substrate. DBP inhibited the oxidation of /S-hydroxybutyrate only in State 3, as indicated in Fig. 5a. DBP did not exhibit an inhibitory effect on respiration in the presence of the substrate only, just as in the case of glutamate oxidation. Effects of Phthalate Esters on the Oxygen Uptake by Sonicated Mitochondria in the Presence of Succinate or NADH— Phthalate esters inhibited the oxidation of succinate, and the
50
100
150
( min )
Fig. 8. Time-dependent inhibition by DOP of the NADH oxidase activity of sonicated mitochondria. Aliquots of the incubated mixture were withdrawn at the indicated times and the activity was measured spectrophotometrically. The final concentration of DOP was 0.128 mM. See "MATERIALS AND METHODS."
inhibition pattern depended on the carbon number of the alkyl groups, as observed with intact mitochondria. A difference of inhibition between straight and branched chain alkyl groups was present, as shown in Fig. 6. The dependence of succinate oxidation by sonicated mitochondria on the concentration of DBP, which was the most potent inhibitor, was also examined. It was found that the inhibition by 3 mM DBP was nearly complete, as in the case of State 3 oxygen uptake by intact mitochondria, and the concentration giving 50% inhibition was 0.18 mM. Figure 7 shows the effect of phthalate es/. Biochem.
EFFECTS OF PHTHALATES ON MITOCHONDRIA
ters on the oxidation of NADH by sonicated mitochondria. DBP inhibited the oxidation of succinate and NADH. The dependence of the inhibition on the carbon number of the alkyl groups was similar to that shown in Fig. 6. The effects of phthalate esters on the NADH oxidase activity of sonicated mitochondria were examined spectrophotometrically. In this case, the degree of inhibition depended on incubation time, probably because a lower concen-
157
tration of sonicated mitochondria was used than in the activity assay by the oxygen electrode method. From Fig. 8, it was found that more than 90 min was necessary for the completion of inhibition when DOP was used. The concentrations of phthalate esters at which NADH oxidase activity fell to 50% were determined. The relation between the logarithm of the concentrations of phthalate esters giving 50% inhibition and the carbon numbers of the alkyl groups is shown in Fig. 9. These results are analogous to those obtained by the oxygen electrode method. Effects of Phthalate Esters on Purified Beef Liver Glutamate Dehydrogenase—Table I shows the effects of different phthalate esters on the activity of beef liver glutamate dehydrogenase. Under the conditions employed, only slight inhibition was observed with DEP, DBP, and DIBP. DISCUSSION
1 2 3 A 5 6 7 8 Carbon Number of Alkyl Group ot Phthalate Esters
Fig. 9. Plot of the logarithm of inhibition constants of phthalate esters in NADH oxidation by sonicated mitochondria against the carbon number of their alkyl groups. The inhibition constants were obtained as the concentrations of phthalate esters giving 50% inhibition after incubation for 90 min (Fig. 8). TABLE I. Effects of phthalate esters on purified beef liver glutamate dehydrogenase. Experimental conditions are given in " MATERIALS AND METHODS." 100 ppm phthalate
Activity
% Inhibition
Dimethyl Diethyl
0.588 0.522
13.0
Di-«-butyl Dihexyl Dioctyl Diisobutyl Diethylhexyl Control
0.504 0.576 0.570 0.540 0.570 0.600
Vol. 79, No. 1, 1976
2.0
16.0 4.0 5.0
10.0 5.0 —
It is considered that there are two kinds of inhibition by phthalate esters of oxygen uptake by intact mitochondria. One occurs at the succinate oxidation site of the electron transport system. This is shown by the fact that the oxygen uptake in State 4 was inhibited when the substrate was succinate but was not inhibited when it was glutamate or /9-hydroxybutyrate. The other is the inhibition of energy transport which is associated with State 3. This is shown by the fact that the oxygen uptake in State 3 was strongly inhibited by phthalate esters in the presence of either succinate or an NAD-linked substrate, and the rate of oxygen uptake decreased to the level of State 4. However, it still remains to be determined which site phthalate esters act on. In the case of sonicated mitochondria, NADH oxidation as well as succinate oxidation was inhibited by phthalate esters. It might be said that the NADH oxidation site can interact with phthalate esters because of the destruction of the mitochondrial membrane by sonication. The biological effect of the carbon number of the alkyl groups of phthalate esters was investigated by Sugawara (14). In his
158
report, five phthalate esters were tested for effect on the hatching of brine shrimp eggs and the order of toxicity was DBP>DHP> DEP>DOP>DMP. Moreover, Kazuya (75) demonstrated that the order of toxicity of phthalate esters was DBP>DEP>DMP using fibroblast cultures from newborn rat cerebellar tissure. However, it was not determined which chemical or physical feature of the phthalate esters was responsible for their toxicity. The results reported here suggest that the toxicity of phthalate esters cannot be determined by their water solubility (16) alone. Calley et al. ( 77), studying the effect of administration i.v. of three phthalate esters (DMP, DEP, DIBP) on the respiratory rate of the anesthetized rabbit, reported that there was a significant increase in respiratory rate after the administration of a total dose of 100 mg/kg and also that the rate gradually returned to the normal level over a period of about 5 min. This is inconsistent with our result that 0.5—2.0 mM DBP, corresponding to 139-556 mg/kg, strongly inhibited the respiration of rat liver mitochondria. The apparent contradiction to be resolved. The potent inhibitory effect of DBP might be explained in terms of specific interaction between the alkyl group of 4 carbon atoms and the inhibitory site. However the details of the mechanism require further experiments for their elucidation. The author's thanks are due to Prof. Dr. I. Yamazaki, Research Institute of Applied Electricity, Hokkaido University for advice, and to Drs. H. Kanashima and Y. Kinoshita, Hokkaido Institute of Public Health, for their help.
T. OHYAMA
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