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The following effects of mercaptoacetic acid have been reported:

·       potentiation of bradykinin-induced contractions of guinea pig gut and uterus [1];

·       inactivation of hypocalcemic activity of the salivary gland hormone, b-parotin [2];

·       stimulation of guinea pig skin histidase activity [3];

·       inhibition of thyroid iodinating enzyme system (in calf thyroid) in the presence of a hydrogen peroxide-generating system [4];

·       in vitro inhibition of uterine response to oxytocin in rats [5-8];

·       diabetogenic effect [10];

·       reduction of rat hepatic succinoxidase activity [11 -12];

·       reduction of bovine antidiuretic factor activity [13];

·       inhibition of fatty acid oxidation [see the following sections].

 

High-throughput screening

 

Mercaptoacetic acid was evaluated in an in vitro high-throughput screening (HTS) (US EPA, 2017) to evaluate the effects on a target genes like, DNA binding, growth factor (transforming growth factor beta), nuclear receptor (orphan, non-steroidal and steroidal), cell cycle, cyp (cytochrome P450 19A1 and 24A1), hydrolase (ATPase), steroid hormones (androgens, estrogens, glucocorticoids and progestagens), transporter and cell morphology (organelle conformation) (US EPA, 2017). 334 assays were performed. Mercaptoacetic acid induced a positive response in only 2 assays:

 

Assay Endpoint Name

Cell Name

Target Family

Gene Name

ATG_ERE_CIS_up

HepG2

nuclear receptor

estrogen receptor 1

ATG_ERa_TRANS_up

HepG2

nuclear receptor

estrogen receptor 1

 

However, both positive assays were flaged as “ Hit-call potentially confounded by overfitting; Only one conc above baseline, active” and deemed not reliable. Therefore, there is no evidence that mercaptoacetic acid could interfere with the expression of the genes screened.

211 bioassays with 361 bioactivity outcomes were retrieved from the PubChem BioAssay database (NCBI, 2017). Mercaptoacetic acid was inactive in 350 bioassays, inconclusive in 5 bioassays and positive in 2 bioassays (4 assays were unspecified). Mercaptoacetic acid had no activity in cell viability assays, no agonist and antagonist activities on the peroxisome proliferator-activated receptor delta (PPARd) and gamma (PPARg), androgen receptor (AR), estrogen receptor alpha (ER-alpha), farnesoid-X-receptor (FXR) and glucocorticoid receptor (GR) signaling pathways. Mercaptoacetic acid was active in the inhibition of metallo-beta-lactamase IMP-1. The toxicological significance, if any, of this bioassay, is unknown.

 

Mechanism of toxic action

 

The mortality and the signs of systemic toxic observed in the oral acute or repeated dose toxicity studies are primarily linked to the inhibition of the ß-oxidation of fatty acids. This inhibition induced secondary effects like a decrease of blood glucose, liver glycogen content, blood and hepatic ketone bodies and liver acetyl-CoA and an increase of plasma free fatty acids and liver triglycerides and acyl-CoA and an enhancement of hepatic pyruvate. The fatty liver induced by mercaptoacetate was mainly due to an inhibition of acyl-CoA dehydrogenase activity and consequently to a marked depression of the ß-oxidation pathway. Fasted animals appeared to be more sensitive to the toxic effects than non-fasted animals.

 

In the divers acute or repeated dose toxicity studies (OECD # 408, 414, 416 and 421) performed by oral route with mercaptoacetic acid and/or its salts, signs of systemic toxic (including mortality), increase of food consumption and/or perturbation of some biochemical parameters related to the fatty acids oxidation were observed. It is suggested that the most probable mechanism of toxicity was linked to the inhibition of the ß-oxidation of fatty acids as described below.

 

Effects of mercaptoacetate on glycemia

Mercaptoacetate has been proved to have an action on blood glucose regulation (Freeman et al., 1956). Respectively, six hours after i. v. (175 mg/kg bw) or i. p. (150 mg/kg bw) treatments with sodium mercaptoacetate, rabbits and rats presented a significantly reduced blood sugar concentration (> 50%), sometimes leading to death. Since the LD50greatly increases in glucose-treated rats (3.4 to 4.4 fold), it is very likely that animals died from hypoglycaemia. Five hours after i. p. treatment (0.04 ml/g of 5% glucose solution ± 630 mg/kg bw sodium mercaptoacetate), the average liver glycogen found in treated mice was significantly reduced (>70%) indicating glycogenolysis due to mercaptoacetate. There was no significant difference in muscle glycogen of treated and untreated animals (3 mL of 50% glucose solution per os± 300 mg/kg bw sodium mercaptoacetate i. p.). In diabetic rats treated with mercaptoacetate, the water intake, the urine volume and the urinary sugar decreased, suggesting that mercaptoacetate was not acting by increasing insulin secretion. There appears to be a rise then a fall in blood sugar in diabetic rabbits after treatment with mercaptoacetate (300 mg/kg bw i. v.).

 

The effects on plasma glucose concentration after administering sodium mercaptoacetate was evaluated to fasted or non-fasted female rats (Grosdidier, 2011). Two groups of 10 female Sprague-Dawley rats received sodium mercaptoacetate in degassed purified water, once daily for 8 or 15 days, by gavage, at a dose-level of 80 mg/kg/day. A constant dose volume of 5 mL/kg was used. Plasma glucose concentration was measured in each animal before the start of treatment (day -1), 1 hour before daily treatment then every 2 hours after treatment until 10 hours on days 1, 8 and addition, it was measured prior to dosing on days 2 and 9 (i.e.24 hours after dosing on days 1 and 8). The first group of animals was fasted overnight prior to the first day of dosing and fasted again prior to the 8thday of dosing. Due to mortality, the decision was taken to stop this group on day 9 and to sacrifice all remaining animals. The second group of animals was not fasted prior to the first and 8thday of dosing but fasted overnight prior to the 15thday of dosing. On day 15, 1.77 mL/kg bw of a 40% glucose solution (equivalent to 0.71 g/kg bw glucose) was administered by oral gavage 1 hour before treatment and immediately after treatment with sodium mercaptoacetate and then at 1, 3, 5 and 7 hours after treatment. During the treatment period, the animals were checked at least twice daily for mortality and regularly for clinical signs. Body weight and food consumption were recorded twice weekly. Remaining animals were sacrificed on completion of the treatment period (day 16) and a complete macroscopic post-mortem examination was performed. The heart and liver were weighed and the heart, liver and pancreas were preserved although no microscopic examination was performed.

In the 1stgroup, 4/10 females were sacrificed for ethical reasons between days 1 and 8. One female was prematurely sacrificed on day 1, and three other females were prematurely sacrificed on day 8, all at approximately the 8-hour blood sampling time-point. Signs of poor condition (such as hunched posture, loud breathing, coldness to the touch, soiled body areas) were observed in these animals before premature termination, as well as hypoactivity that turned to recumbency and then coma. Two other females were found dead before treatment on day 9 after showing similar clinical signs on day 8. The remaining four females of this group were sacrificed on day 9 after the decision to stop the group. In the 2ndgroup, 2/10 females were prematurely sacrificed, one on day 7, and one on day 9 of treatment due to marked body weight loss and/or clinical signs of poor condition. The remaining animals were kept until the end of the study, on day 16.

On day 1, clinical signs of poor condition were observed in all group 1 animals (animals fasted prior the 1stand the 8thadministration), this included hunched posture, piloerection, half-closed eyes, mydriasis, tremors, hypoactivity, cold to the touch, prostration and abdominal breathing. From days 2 to 4 the clinical condition improved markedly and no clinical signs were observed on day 5. Generally from day 6 until the study stopped on day 9, constant chewing movement was observed in all animals. On day 8, after overnight fasting, the same marked clinical signs as on day 1 were observed after treatment and were accompanied by other signs of poor condition (among which: ventral recumbency, comatose, reddish-colored urine, abnormal vocalization, immobilization of hindlimbs). On day 9, before premature sacrifice, all the remaining animals (four females) mainly had hunched posture and piloerection. No clinical signs were observed on day the animals of group 2 (animals not fasted prior the 1stand 8thadministration and fasted prior the 15thadministration). However, from day 3 some of the animals started having clinical signs (hunched posture, piloerection, thin appearance). Generally from day 6, all animals had constant chewing movement (this was sporadically seen until the end of the study) and other signs such as hunched posture, piloerection, dyspnea, thin appearance and/or thinning of hair were observed in most of the animals. From day 9, more clinical signs were observed in the animals (tremors, abdominal breathing, widespread hindlimbs, locomotor difficulties, walking on tiptoe). On day 15, following overnight fasting of these animals and in spite of oral administration of a 40% glucose solution, clinical signs of poor condition were observed in all animals.

Almost all animals from group 1 lost body weight between day -1 and day 3 which was likely to have been due to the fasting combined with treatment. From day 3 to day 7, all animals gained body weight. The group 2 animals had low or no mean body weight gains for the first 10 days of treatment, except for 1/10 female that actually lost 21% of its starting weight. Body weight gains improved between day 10 and day 14.

Mean food consumption of group 1 animals was initially low (day -1 to day 3) but was higher during the second part of the week (day 3 to day 7). The mean food consumption of group 2 animals was low until day 10 of treatment, correlating with the low body weight gains.

On day -1 as well as on days 2 and 9 before dosing, the mean blood glucose concentrations of the groups 1 and 2 animals were similar. This confirmed that both groups started with similar basal conditions. On days 1 and 8, the mean blood glucose level of group 2 animals tended to minimally decrease from the time-point 2 hours until the 6 hours time-point, and then increased between 6 and 10 hours post-dose, so that the last time-point was similar to the pre-dose value. The mean blood glucose concentration of group 1 animals also decreased over time but was lower than that of group 2 animals at the time-points 4 hours and 6 hours after dosing, to finally return to pre-dose values at 10 hours post-dose.

On day 15, when the group 2 animals were subjected to a fasting period before treatment, with co-administration of a glucose solution at regular intervals, the glucose level of the animals was always higher than the baseline and was relatively constant over time.

 

Thioglycolate is known to inhibit the mitochondrial beta-oxidation of fatty acids in liver resulting in a greater conversion of the latter into triglycerides that accumulated in the liver, as a result, ketogenesis was inhibited (Bauchéet al., 1977, 1981, 1982 and 1983). A former investigor (Freeman et al., 1956) have described at 60% decrease of the blood glucose level 5 to 6 hours after the i.p. injection of 150 mg/kg bw sodium mercaptoacetate to fasted rats and when male rats were given increasing doses of sodium mercaptoacetate i.p. plus one ml. of a 50% solution of glucose i.p initially and every two hours for six hours, the LD50was raised from 126 ± 9 mg/kg. to 426 ± 54 mg/kg. These investigations indicated that the effect on the blood glucose level was a key factor in the toxicity of sodium mercaptoacetate. The present investigations showed a trend toward decreased blood glucose level 4 to 6 hours after administration of sodium mercaptoacetate both with or without fasting. Fasting worsened this trend, with episodes of severe hypoglycaemia. When fasting was accompanied by an oral administration of glucose, the blood glucose concentration remained constant and high. 

As mortality/poor condition was mainly seen when treatment was administered after fasting (days 1 and 8), this suggested a possible contribution of hypoglycaemia to the clinical picture. Nevertheless, when the blood glucose was maintained at high level by oral administration of glucose, signs of poor condition occurred in all fasted animals, and thus other parameters than hypoglycaemia may have contributed to the worsening of clinical signs in these animals.

When compared to historical control animal organ weight data, there were higher absolute and relative-to-body liver weights in group 1 and 2 animals.

No macroscopic findings could be clearly attributed to treatment in prematurely sacrificed or found dead animals. Liver enlargement was noted in one group 1 female sacrificed on day 9, along with tan discoloration. Tan discoloration in the liver was also observed in one group 2 female sacrificed on daylight of the liver weights, these gross findings were possibly treatment-related. Dilated urinary bladders with red contents were noted in two out of six prematurely dead group 1 females. A relationship to treatment with sodium mercaptoacetate could not be excluded.

In conclusion, signs of poor condition were observed in all animals previously fasted after day 1 administration. Animals partially recovered when treatment was given without fasting, but after the day 8 administration preceded by fasting, signs of poor condition were seen again and that led to several unscheduled deaths and finally the study was stopped on day 9 for the fasted group. In the fasted group, body weight was affected until day 3 only, as well as food consumption. Signs of poor condition were observed in the non-fasted group from generally day 3. More clinical signs were then observed from day 9. On day 15, when test item and glucose were administered after fasting, signs of poor condition were seen in all animals, but blood glucose was maintained at normal levels. In these animals, body weight gain was low over the study, as well as food consumption. In all animals, at post-mortem examination, findings were seen in the liver: higher mean absolute and relative-to-body liver weights were noted in both groups, as well as liver enlargement and/or tan discoloration in one single animal from each group. Dilated urinary bladders with red contents were noted in two out of six prematurely dead fasted females. A relationship to treatment with sodium mercaptoacetate could not be excluded. As mortality/poor condition was mainly seen in the fasted group, when treatment was administered after fasting (days 1 and 8), this suggested a possible contribution of hypoglycaemia to the clinical picture. Nevertheless, when the blood glucose was maintained at high level by oral administration of glucose, signs of poor condition but no mortality occurred in all fasted animals, and thus other parameters than hypoglycaemia may have contributed to the worsening of the condition of these animals.

 

Effects of mercaptoacetate on the fatty acid oxidation and liver enzyme activity

On the basis of the published results (Nordmann and Nordmann, 1971; Sabourault et al.,1976; Sabourault et al.,1979; Bauché et al.,1981; Bauché et al.,1982; Bauché et al.,1983.; Sabbagh et al.,1985; Schulz, 1987), there is no doubt that mercaptoacetate inhibited the hepatic-ßoxidation of fatty acids resulting in a greater conversion into triglycerides in the liver. As a result, ketogenesis was inhibited.

 

Intraperitoneal administrations (31 mg/kg bw and 15 mg/kg bw, 3 hours later) of 2-mercaptoethanol (which is oxidized to mercaptoacetate) causes a fatty liver, as shown by the highly significant (p<0.01) increase of liver triglycerides (2.2x), accompanied by a considerable increase of plasma free fatty acids (2.4x) and by remarkable decreases in blood ketone bodies (acetoacetate –73% and ß-hydroxybutyrate –90%), 3 hours after the 2ndinjection. A very significant (p<0.01) decrease of blood glucose (-35%) and liver glycogen (-70%) content occurs at the same time (Nordmann and Nordmann, 1971).

It appears that mercaptoacetate administered to female Wistar rats (45 mg/kg bw, i. p.) induced an increase of hepatic triacylglycerol (2.7x at 3h and 13.7x at 24h) and blood free fatty acids (3.9x at 3h and no effects at 24h), a decrease of blood triacylglycerol (-30% at 3h and –48% at 24h) and phospholipids (-43% at 3 h and –18% at 24h) as well as a reduction in the hepatic ketone body level (acetoacetate –52% at 3h and ß-hydroxybutyrate –75% at 3h and –42% at 24h). The large and early increase of blood free fatty acids reflects most probably an enhanced peripheral fat mobilization, which is an important factor in the pathogenesis of fatty liver (Sabourault et al., 1976).

After mercaptoacetate administration (45 mg/kg bw i. p.) the hepatic levels of free CoA-SH and acetyl-CoA were markedly decreased, falling to c. a. 20% of the control values. At the same time, on the contrary, the hepatic acyl-CoA level was increased (+120%), an effect which did not completely balance, however, the reduction in free CoA-SH and acetyl-CoA concentrations. Moreover, 2-mercaptoacetate treatments induced both a dramatic increase (> 15x) of the hepatic pyruvate level and a significant reduction (-40%) of the blood glucose level (Sabourault et al., 1979). The increase in hepatic pyruvate level could result from a direct/indirect inhibition of the mitochondrial utilization of pyruvate by mercaptoacetate.

Indeed, it has been shown, using rat hepatic mitochondria, that mercaptoacetate could be a substrate for acetyl-CoA synthase, following an ATP-dependent activation, and that the resulting compound, 2-mercaptoacetyl-CoA could inhibit non-specifically the fatty acyl-CoA dehydrogenases (long-chain general and short-chain acyl-CoAdehydrogenases) as well as the branched-chain acyl-CoA dehydrogenase, namely isovaleryl-CoA (Bauché et al., 1982 and Bauché et al., 1983; Sabbagh et al., 1985).

Mercaptoacetate injected parenterally or administered by gavage, significantly depressed hepatic succinoxidase (SO) in rats, mice and rabbits, the male displaying a higher sensitivity than the adult female.

In vitro, mercaptoacetate was without effect on cytochrome oxidase and diaphorase activities but depressed NADH cytochrome-c-reductase activity. In vivo, mercaptoacetate was without action on the SO activity in spleen and brain but was inhibited in liver and kidneys of male rats. The activities of the hepatic xanthine oxidase, D-amino acid oxidase, malic oxidase, LDH and alcohol deshydrogenase as well as the activities of the renal LDH and alcohol deshydrogenase were not affected. The feeding of diets supplemented with excessive amounts of amino acids prior to the injection of mercaptoacetate, protected against the action of the thiol (Bakshy and Gershbein, 1971 and Bakshy and Gershbein, 1973).

 

Effects of mercaptoacetate on food consumption

The effect of mercaptoacetate on food consumption seems to depend on the age, the strain, the nutritional status and on the nature of the dietary fat.

 

Several experiments have shown that mercaptoacetate increased the food intake in medium- or high-fat fed animals but not in low-fat fed animals (Scharrer and Langhans, 1986). This increase was essentially due to the reduced interval between meals rather than the increase of the meals size or duration (Langhans and Scharrer, 1987). The inhibition exerted by mercaptoacetate on the ß-oxidation of the fatty acids was considered to be the earliest metabolic signal modifying independent ingestion in rats (increase of the plasma free fatty acids, decrease of plasma ß-hydroxybutyrate confirmed the inhibition). A long-term feeding-inhibitory effect could also be observed after mercaptoacetate administrations, probably mediated by a different mechanism than its feeding-stimulatory effect (Brand tet al., 2006).

Several hypothesis were issued to explain the control of the food intake by the ß-oxidation:

·The decrease in reduced cofactors (Nordmann and Nordmann, 1971; Scharrer and Langhans, 1986) increases the food intake.

·The inhibition of the ß-oxidation decreased the membrane potential in liver cell, which modulate the afferent vagal activity, stimulating the food intake (Boutellier et al., 1999). Mercaptoacetate-induced enhanced feeding in rats given fat-enriched diet does not depend on a stronger hepatic and/or celiac vagal afferent response than rats given a low-fat diet (Randich et al., 2002). In addition, the release of ketone bodies could independently act on the afferent vagal activity.

·The NMDA receptors and the neurotransmitter glutamate may be involved in processing these mecanoreceptive signals (Duva et al., 2005).

·Some results suggest that mercaptoacetate elicits a feeding inhibitory effect in fasted rats, which could be due to an increased ß-adrenergic activity. Hypothermia, increased plasma free fatty acids levels and eventually disturbances in glucose metabolism may have contributed (Brandt et al., 2006).

·Glucose deprivation does not contribute to feeding elicited by mercaptoacetate-induced inhibition of fatty acid oxidation in rats fed a carbohydrate-free, high-fat diet (Del Prete et al., 2001).

 Experiments performed on food intake in developing rats (Swithers et al., 2000, Swithers et al., 2001), in adequation with effects observed in adult rats, show that the action of mercaptoacetate, by blockade of fatty acid oxidation, stimulates independent ingestion and not suckling or water intake. This stimulation does not work on younger animals, 9-weeks aged old or lower. This is probably due to a compensatory system present at this period of development. In addition, mercaptoacetate has a short-term action (shorter than to the duration of ß-oxidation inhibition) probably antagonized later by the disturbances of metabolite homeostasis resulting from the impairment of fatty acid oxidation. At high dose-levels of mercaptoacetate, inhibition of the food intake and gastric emptying were observed.

Additional information

Additional references:

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2. Namba S. Betaparotin. VI. The effect of various chemical reagents and proteolytic enzymes on beta-protein. Endocrinol Jpn. 1966;12:268-276.

3. Anglin JH, Jones DH, Bever AT, Mevertt MA. The effect of ultraviolet light and thiol compunds on guinea pig skin histidase. J Invest Dermatol. 1966;46:34-39.

4. Degroot LJ. Stimulation and inhibition of thyroid iodinating enzyme systems. Biochem Biophy Acta. 1967;13:364-374.

5. Taira N, Marshall JM. Action of oxytocin antagonists on electrical and mechanical activity of the uterus. Am J Physiol. 1967;212:725-731.

6. Anderson WG, Pihlaja P, Miller JW. Antagonism of the uterotonic action of oxytocin in vitro. J Pharmacol Exp Ther. 1975;192:399-407.

7. Martin PJ and Child HO. Effects of thiols on oxytocin and vasopressin receptors. Nature. 1962, 196:382-383.

8. Martin PJ and Schild HO. Antagonism of Disulphide Polypeptides by Thiol.Brit. J. Pharmacol. 1965, 25, 418-431.

9. Holder FC. Thioglycolate de sodium et hormones hypothalamo-neurohypophysaires.Experientia. 1973, 29(4):487–488

10. Inoue Y. Alloxan-like action of some sulfhydryl inhibitors on glucose tolerance in rabbits. Endocrinol Jpn. 1968; 15:1-7.

11. Bakshy S, Gershbein LL. In vivo depression of hepatic succinoxidase by sulfhydryl compounds. Biochem Biophys Res Commun. 1971;42:765-771.

12. Bakshy S, Gershbein LL. Affect of sulfur compound on various sulfhydryl-dependent enzyme systems. Arch Int Pharmacodyn Ther. 1973;201:77-89.

10. Rudman D, Chawla RK. Antidiuretic peptide in mammalian chroid plexux. Am J Physiol. 1976;230:50-55.