Significance of sulfhydryl compounds in the manifestation of fluoroacetate toxicity to the rat, brush-tailed possum, woylie and western grey kangaroo

Levels of citrate in kidneys and livers of rats with normal glutathione levels increased 6.8 and 1.7-fold respectively 2 h after dosing with 1.5 mg of compound 1080 (= 95% sodium fluoroacetate) per kilogram body weight. In animals with liver glutathione levels 15% of normal, increases in plasma and liver citrate levels after dosing with fluoroacetate were significantly greater than those of control animals. Cysteamine and N-acetylcysteine, like glutathione, partially protected aconitate hydratase from fluorocitrate inhibition in rat liver preparations but were unable to replace glutathione as a substrate for the defluorination of fluoroacetate in vitro. N-Acetylcysteine did not diminish plasma citrate levels of glutathione-deficient rats dosed with fluoroacetate, while cysteamine inhibited the rate of in vivo defluorination in glutathione-deficient brush-tailed possums. It is suggested that non-physiological sulfhydryl compounds are ineffective antidotes to fluoroacetate intoxication in vivo. The in vivo defluorination patterns of four mammal species with differing sensitivities to fluoroacetate did not indicate a direct relationship between tolerance and rate of defluorination and it is also suggested that a high level of activity of the glutathione-S-transferase responsible for the defluorination of fluoroacetate is not the major mechanism for circumventing fluoroacetate toxicity in resistant mammals.     

Inhibitory effects of cysteamine on neuroendocrine function

The action of cysteamine on anterior pituitary hormone secretion was studied in vivo using conscious, freely moving male rats and in vitro using anterior pituitary cells in monolayer culture. Administration of 500 micrograms cysteamine into the lateral cerebral ventricles of normal rats caused the complete inhibition of pulsatile GH secretion for a minimum of 6 h. This treatment also significantly decreased plasma concentrations of LH for at least 6 h in orchiectomized rat, TSH in short-term (0.5 month) thyroidectomized rats, and PRL in long-term (6 months) thyroidectomized rats. The in vivo stimulation of GH, LH, TSH and PRL with their respective releasing hormones 60 min after administration of cysteamine was not different from the response observed in rats pretreated with saline except for PRL where cysteamine pretreatment significantly inhibited the expected PRL increase. In vitro, 1 mM cysteamine decreased basal and TRH stimulated PRL release while not affecting basal or stimulated GH, LH, TSH and ACTH secretion. These data demonstrate the dramatic and wide-ranging effects of cysteamine on anterior pituitary hormone secretion. This action appears to be mediated through hypothalamic pathways for GH, LH and TSH and through a pituitary pathway for PRL.     

Cysteamine suppresses invasion, metastasis and prolongs survival by inhibiting matrix metalloproteinases in a mouse model of human pancreatic cancer

Background

Cysteamine, an anti-oxidant aminothiol, is the treatment of choice for nephropathic cystinosis, a rare lysosomal storage disease. Cysteamine is a chemo-sensitization and radioprotection agent and its antitumor effects have been investigated in various tumor cell lines and chemical induced carcinogenesis. Here, we investigated whether cysteamine has anti-tumor and anti-metastatic effects in transplantable human pancreatic cancer, an aggressive metastatic disease.

Methodology/Principal Findings

Cysteamine''s anti-invasion effects were studied by matrigel invasion and cell migration assays in 10 pancreatic cancer cell lines. To study mechanism of action, we examined cell viability and matrix metalloproteinases (MMPs) activity in the cysteamine-treated cells. We also examined cysteamine''s anti-metastasis effect in two orthotopic murine models of human pancreatic cancer by measuring peritoneal metastasis and survival of animals. Cysteamine inhibited both migration and invasion of all ten pancreatic cancer cell lines at concentrations (<25 mM) that caused no toxicity to cells. It significantly decreased MMPs activity (IC ₅₀ 38–460 µM) and zymographic gelatinase activity in a dose dependent manner in vitro and in vivo; while mRNA and protein levels of MMP-9, MMP-12 and MMP-14 were slightly increased using the highest cysteamine concentration. In vivo, cysteamine significantly decreased metastasis in two established pancreatic tumor models, although it did not affect the size of primary tumors. Additionally, cysteamine prolonged survival of mice in a dose-dependent manner without causing any toxicity. Similar to the in vitro results, MMP activity was significantly decreased in animal tumors treated with cysteamine. Cysteamine had no clinical or preclinical adverse effects in the host even at the highest dose.

Conclusions/Significance

Our results suggest that cysteamine, an agent with a proven safety profile, may be useful for inhibition of metastasis and prolonging the survival of a host with pancreatic cancer.     

Dose-related effects of cysteamine treatment on hypothalamic and striatal dopamine, noradrenaline and serotonin neurotransmission

The dose-related effects of cysteamine treatment on hypothalamic and striatal neurotransmission were investigated. Cysteamine pretreatment with a dose of 150 mg/kg slightly increased the dopamine, and markedly decreased the noradrenaline, content of the hypothalamus in a dose-related manner. The serotonin levels of the hypothalamus and striatum were not affected. Cysteamine pretreatment with a higher dose (300 mg/kg sc) slightly increased the uptake of noradrenaline into hypothalamic slices. The drug did not influence dopamine and serotonin uptake into hypothalamus and striatal slices. These results suggest that cysteamine decreases rather selectively the noradrenaline content of the hypothalamus.     

Mechanisms involved in the depleting effect of cysteamine on pancreatic somatostatin

We investigated the effects of cysteamine on the pancreatic islet hormones and found that pancreatic somatostatin contents depleted 60 min after the oral administration of cysteamine (300 mg/kg) to rats, yet the insulin and glucagon contents remained unchanged. When pancreatic islets isolated by collagenase digestion were incubated for 60 min in Krebs-Ringer bicarbonate buffer containing 0.1, 1, or 10 mM cysteamine, cysteamine dose-dependently decreased the somatostatin content, however, only a high concentration (10 mM) decreased the insulin level, and cysteamine exerted no effect on the glucagon content. The islet hormones (synthetic somatostatin-14, synthetic somatostatin-28, extracted pork insulin and extracted pork glucagon) were incubated for 60 min with cysteamine (0.1, 1, or 10 mM) and somatostatin-14 was found to be markedly decreased by 1 mM cysteamine. Pork insulin but not pork glucagon was dose-dependently decreased by 0.1-10 mM cysteamine. Cysteamine, 0.1-1 mM, did not interfere with the radio-immunoassay system for somatostatin or insulin, although 10 mM cysteamine did so. This compound exerted no effect on the radioimmunoassay system for glucagon. Our studies support earlier findings that cysteamine administered to experimental animals plays a role of relatively specific depletor of somatostatin. The possibility that the depletion of somatostatin is in part due to the remarkable sensitivity of the intracellular compartments of the D cells to the drug and in part due to the remarkable sensitivity of the molecular structure of somatostatin has to be considered.     

The effect of muscarinic and beta-adrenergic blockade on cysteamine-induced gastrin secretion by the isolated perfused rat stomach

Cysteamine-induced duodenal ulceration in rats is accompanied by increased circulating gastrin. Although cysteamine appears to exert a direct action on the gastrin cell some groups have provided evidence for an involvement of the autonomic nervous system. The current experiments were performed to determine whether beta-adrenergic or cholinergic (muscarinic) pathways are involved in the acute effect of cysteamine on gastrin secretion in the isolated perfused rat stomach. Cysteamine (1 mM) increased gastrin (IRG) secretion to a maximum ranging between 100% and 192% above basal. A cysteamine concentration of 5mM resulted in peak levels ranging between 150% and 1050% above basal. Addition of atropine or propranalol did not influence the responses obtained. The present results, therefore, do not support a role for either cholinergic or beta-adrenergic pathways in cysteamine-induced gastrin release at the level of the stomach and suggest that in vivo such autonomic effects are mediated extrinsically.     

Monocyte aryl hydrocarbon hydroxylase (AHH) activity mimics Kupffer cell and hepatocyte AHH activity in an animal model of liver disease.

We have previously reported that monocyte aryl hydrocarbon hydroxylase (AHH) activity is depressed in patients with liver disease and is decreased more in cirrhosis than in early stage liver disease. To determine if monocyte AHH activity reflects liver AHH activity, we studied an animal model of cirrhosis, i.e., yellow phosphorus induced cirrhosis in the pig. AHH activity was detectable in monocytes isolated from peripheral blood of normal pigs (0.32 +/- 0.13 nmol.mg-1 P.h-1, n = 11) and was comparable to the level of AHH activity in hepatic Kupffer cells isolated from wedge or needle biopsies of livers of normal pigs (0.38 +/- 0.21, n = 7). The AHH level in pig Kupffer cells was approximately 10% of the AHH level in hepatocytes and microsomes. To induce liver disease, pigs were administered yellow phosphorus (0.6 mg/kg) 5 days per week for 16 weeks. At 4 weeks of treatment, monocyte AHH activity was not different from control and liver histology was normal. Depression of monocyte AHH activity was evident at 8 weeks of treatment when liver fibrosis was seen histologically. At 12 weeks of treatment when histology revealed extensive liver fibrosis and collagen levels were elevated, the level of monocyte AHH activity was decreased 67% compared with controls. Similar changes were observed at 12 weeks in Kupffer cell AHH activity (86% decrease) and hepatocyte AHH activity (70% decrease) compared with controls. These results suggest that monocyte AHH activity reflects liver AHH activity and may be a good indicator of change in liver enzyme function in liver disease in the pig model of cirrhosis.     

Cysteamine exerts multiple effects on the endocrine cells of the rat pancreas

We have studied the effects by cysteamine in vitro and in vivo on hormone production and islet cell metabolism in isolated pancreatic islets and perfused pancreas of the rat. In isolated islets, cysteamine dose-dependently depleted somatostatin immunoreactivity by 50% after 60 min exposure to 1 mmol/l of the compound. This effect appeared to be independent of interaction of the drug with secretion of somatostatin from the pancreatic D-cells. Cysteamine, however, interacted acutely not only with the D-cells, but also markedly suppressed glucose-induced insulin release. Moreover, cysteamine inhibited islet glucose oxidation, an effect which reflects interference with the metabolism mainly of the B-cells. The effect of cysteamine on glucose-induced insulin release was prolonged, since it was still observed in the isolated rat pancreas perfused 24 h after in vivo treatment with cysteamine. In contrast to the effects on glucose-induced insulin release, the response to glibenclamide remained unaffected by a previous exposure to cysteamine in vivo. However, both glucose- and glibenclamide-induced somatostatin secretion was reduced by 50%, whereas basal glucagon secretion was significantly enhanced in pancreata from cysteamine-treated rats vs. control rats. We conclude that (1) cysteamine does not specifically affect the D-cells of the islets, and (2) the multiple effects by cysteamine on islet cell function, particularly on B-cell metabolism and secretion, renders the compound unsuitable for the study of paracrine interactions in the islets.     

Myeloperoxidase-oxidase oxidation of cysteamine.

Cysteamine oxidation was shown to be catalysed by nanomolar concentrations of myeloperoxidase in a peroxidase-oxidase reaction, i.e. an O2-consuming oxidation of a compound catalysed by peroxidase without H2O2 addition. When auto-oxidation of the thiol was prevented by the metal-ion chelator diethylenetriaminepenta-acetic acid, native, but not heat-inactivated, myeloperoxidase induced changes in the u.v.-light-absorption spectrum of cysteamine. These changes were consistent with disulphide (cystamine) formation. Concomitantly, O2 was consumed and superoxide radical anion formation could be detected by Nitro Blue Tetrazolium reduction. Both superoxide dismutase and catalase inhibited the reaction, whereas the hydroxyl-radical scavengers mannitol and ethanol did not. O2 consumption increased with increasing pH (between pH 6.0 and 8.0), and 50% inhibition was exhibited by about 3 mM-NaCl at pH 7.0 and by about 100 mM-NaCl at pH 8.0. Cysteamine was about 5 times as active (in terms of increased O2 consumption at pH 7.5) as the previously reported peroxidase-oxidase substrates NADPH, dihydroxyfumaric acid and indol-3-ylacetic acid. A possible reaction pathway for the myeloperoxidase-oxidase oxidation of cysteamine is discussed. These results indicate that cysteamine is a very useful substrate for studies on myeloperoxidase-oxidase activity.     

Effects of clofibrate on the main regulatory enzymes of cholesterogenesis

The in vivo effect of clofibrate on the main regulatory enzymes of cholesterogenesis has been comparatively studied for the first time in chick liver and brain. 3-Hydroxy-3-methylglutaryl-CoA reductase and mevalonate 5-pyrophosphate decarboxylase from chick liver were significantly inhibited by this hypocholesterolenic drug, while mevalonate kinase and mevalonate 5-phosphate kinase were not affected. No enzyme from chick brain was significantly inhibited by the in vivo treatment. However, both liver and brain reductase activity was inhibited in vitro by clofibrate, inhibition that was progressive with increasing concentrations (1.25-5.00 mM) of drug.     

In vitro and in vivo inhibition of pulmonary cytochrome P450 activities by piperine, a major ingredient of piper species

In vitro and in vivo modulation of drug metabolizing enzymes by piperine was investigated in microsomes of rats and guinea pigs. In vitro piperine caused concentration related inhibition (50% at 100 microM) of arylhydrocarbon hydroxylase (AHH) and 7-ethoxycourmarin deethylase (7ECDE) activities, which were comparable in control and 3-methylcholanthrene (3MC) treated rats. In guinea pig microsomes however, piperine caused strong inhibition at lower concentrations (35% at 10 microM) and relatively much lesser inhibition with further increase in piperine concentrations. A Dixon plot of the kinetic data of both AHH and 7ECDE indicated noncompetitive inhibition with a Ki of approx. 100 microM. In vivo, piperine given at a dose of 25 mg/kg body wt to rats caused a maximal inhibition at 1 hr of both the enzymes, while only AHH returned to normal value within 4 hr. Similarly, upon daily treatment of piperine (15 mg/kg body wt) to rats for 7 days, 7ECDE was consistently inhibited, while AHH showed faster recovery. Piperine thus appeared to cause differential inhibition of two forms of cytochrome P450 and thus would accordingly affect the steady-state level of those drugs metabolized by these pulmonary forms of cytochromes P450.     

Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender

The aim of this study was to evaluate the effects of ergothioneine and cysteamine as antioxidant supplements in a soybean lecithin extender for freezing ram semen. Twenty-four ejaculates were collected from four rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and cysteamine or ergothioneine (2, 4, 6 or 8 mM). Motility by CASA, viability, plasma membrane functionality (HOS test), total abnormality, lipid peroxidation, glutathione peroxidase (GPx) activity and capacitation status (CTC staining) were assessed after thawing. Using 6 mM of either antioxidant improved total motility. Cysteamine at 6 mM and ergothioneine at 4 and 6 mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). Both antioxidants improved membrane functionality significantly, except at 8 mM. Progressive motility, kinematic parameters, GPx activity, capacitation status and sperm abnormalities were not influenced by the antioxidant supplements. In conclusion, cysteamine at 6 mM and ergothioneine at 4 or 6 mM seem to improve the post-thawing quality of ram semen cryopreserved in a soybean lecithin extender.     

Differences in action of topical and systemic cysteamine on gastric blood flow,gastric acid secretion and gastric ulceration in the rat

The effect of cysteamine on gastric blood flow and on the indomethacin-induced gastric mucosal damage was studied. In anesthetized rats, cysteamine (280 mg/kg) given subcutaneously (sc) decreased gastric blood flow measured by the laser Doppler flowmetry technique. In contrast, cysteamine (1–60 mg/ml) applied topically to the serosal surface of the stomach evoked a concentration-dependent and long-lasting increase in gastric blood flow. At 60 mg/ml, cysteamine increased blood flow by 166.8 ± 26.1% of predrug control value. Pretreatment with indomethacin (20 mg/kg, sc), intravenous (iv) atropine (1 mg/kg), propranolol (1 mg/kg, iv), combined H₁ and H₂-blockade or bilateral cervical vagotomy alone or combined with iv guanethidine (8 mg/kg), or pretreatment with the capsaicin analogue resiniferatoxin did not reduce the vasodilator response to cysteamine. The vasodilator response to topical capsaicin, was not reduced after sc cysteamine (280 mg/kg) pretreatment. In conscious pylorus-ligated rats, sc cysteamine (100 or 280 mg/kg) given simultaneously with indomethacin inhibited gastric acid output but had variable effects on the indomethacin-induced gastric mucosal damage. Cysteamine (100 or 280 mg/kg) administered sc 4 h prior to indomethacin enhanced gastric injury by sc indomethacin, but did not prevent the gastroprotective action of capsaicin. In contrast, orally administered cysteamine (60 mg/ml) reduced gastric injury induced by sc indomethacin plus intragastric HCl. These data provide the first evidence for the effect of cysteamine on gastric microcirculation in the rat and suggest a direct vasodilator effect for topical cysteamine. The microvascular effects of cysteamine are largely responsible for the different effects of this agent on experimental gastric injury.     

Ratliver cells in culture: Effect of storage,long-term culture,and transformation on some enzyme levels

Summary Aryl hydrocarbon hydroxylase (AHH) and tyrosine aminotransferase (TAT) activities were determined in rat liver cell lines after frozen storage, long-term culture, and transformation in vitro. Levels of AHH activity after 17 months in frozen storage were comparable to levels prior to freezing. During long-term culture the AHH levels of the cell lines tended to decrease. Transformed lines had variable levels of AHH activity. Cell lines retained measurable TAT activity following long-term culture and frozen storage. TAT activity of transformed cells was comparable to that of normal lines. Prolonged frozen storage did not induce transformation up to one year.     

Inhibition of hepatic aldehyde dehydrogenases in the rat by calcium carbimide (calcium cyanamide)

Oral administration of 7.0 mg/kg calcium carbimide (calcium cyanamide, CC) to the rat produced differential inhibition of hepatic aldehyde dehydrogenase (ALDH) isozymes, as indicated by the time-course profiles of enzyme activity. The low-Km mitochondrial ALDH was most susceptible to inhibition following CC administration, with complete inhibition occurring at 0.5 h and return to control activity at 96 h. The low-Km cytosolic and high-Km mitochondrial, cytosolic, and microsomal ALDH isozymes were inhibited to a lesser degree and (or) for a shorter duration compared with the mitochondrial low-Km enzyme. The time course of carbimide, the hydrolytic product of CC, was determined in plasma following oral administration of 7.0 mg/kg CC to the rat. The maximum plasma carbimide concentration (102 ng/mL) occurred at 1 h and the apparent elimination half-life in plasma was 1.5 h. Carbimide was not measurable in the liver during the 6.5 h time interval when carbimide was present in the plasma. There were negative, linear correlations between plasma carbimide concentration and hepatic low-Km mitochondrial, low-Km cytosolic, and high-Km microsomal ALDH activities. In vitro studies demonstrated that carbimide, at concentrations obtained in plasma following oral CC administration, produced only 19% inhibition of low-Km mitochondrial ALDH and no inhibition of low-Km cytosolic and high-Km microsomal ALDH isozymes. These data demonstrate that carbimide, itself, is not primarily responsible for hepatic ALDH inhibition in vivo following oral CC administration. It would appear that carbimide must undergo metabolic conversion in vivo to inhibit hepatic ALDH enzymes, which is supported by the observation of no measurable carbimide in the liver when ALDH was maximally inhibited following oral CC administration.     

Effects of cysteamine, fsh and estradiol-17beta on in vitro maturation of porcine oocytes

Porcine cumulus-oocyte complexes (COCs) were cultured for 48 h with addition or absence of exogenous estradiol-17beta (E2; 1 microg/mL) in the maturation medium (mM199). The medium was supplemented with sodium pyruvate (0.1 mg/mL), 10% (v/v) FCS, various concentrations of FSH (0, 1 and 10 microg/mL) and with or without cysteamine (150 microM). When supplemented with E2, cysteamine enhanced the rates of germinal vesicle breakdown (GVBD) and maturation to metaphase-II (M-II) in COCs cultured in the medium with 0 and 1 microg/mL FSH (P<0.05). Among COCs cultured with FSH, oocytes cultured with 1 microg/mL FSH and E2 but without cysteamine showed the lowest rates of GVBD and M-II. The rates were, however, significantly increased when cysteamine was added to the same medium or by increasing FSH concentration to 10 microg/mL in the maturation medium. E2 significantly inhibited the rates of GVBD and M-II in COCs cultured without FSH and cysteamine (a group of oocytes with spontaneous maturation). When COCs were cultured in TCM 199 with 1 or 10 microg/mL FSH, with or without E2 (1 microg/mL) and fertilized in vitro, the rates of male pronucleus formation were not increased by increasing FSH concentration, but the addition of cysteamine to the maturation medium significantly enhanced the rates in the same FSH treatment. The results indicate that E2 inhibits spontaneous GVBD and maturation to M-II of porcine oocytes and that a low concentration of FSH (1 microg/mL) is not sufficient to induce full nuclear maturation, compared with 10 microg/mL FSH, but that it can complete nuclear maturation with cysteamine and E2. However, the cytoplasmic maturation is promoted only by the addition of cysteamine in the medium.     

In vitro and in vivo effects of naringin on cytochrome P450-dependent monooxygenase in mouse liver

In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid. In vitro addition of naringin up to 500 microM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes. In contrast, the aglycone naringenin at 300 to 500 microM decreased AHH activity by 50% to 60%. Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 microM. Naringenin at 100 microM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as beta-galactosidase activity was reduced to a level similar to the control value. Administration of a liquid diet containing 10 mg/ml naringin for 7 days caused 38% and 49% decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively. In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone. Microsomal P450 and cytochrome b5 contents and NADPH-P450 reductase activity were not affected. Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38%. These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo. These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2.     

Interrelation between the analgesic and ulcerogenic action of cysteamine

Interrelationship was studied between the influence of cysteamine on pain threshold and ulcerogenic effect on the duodenum. Cysteamine (350 mg/kg) induced analgesia in mice which was prevented by naloxone (1.5 mg/kg). In rats, cysteamine produced duodenal ulcers with concomitant analgesia. The intensity of ulceration was higher in animals with lower basal pain threshold. The correlation between central and peripheral effects of endogenous opioids in the development of experimental duodenal ulcers is discussed.     

Rat liver cells in culture: effect of storage, long-term culture, and transformation on some enzyme levels.

Aryl hydrocarbon hydroxylase (AHH) and tyrosine aminotransferase (TAT) activities were determined in rat liver cell lines after frozen storage, long-term culture, and transformation in vitro. Levels of AHH activity after 17 months in frozen storage were comparable to levels prior to freezing. During long-term culture the AHH levels of the cell lines tended to decrease. Transformed lines had variable levels of AHH activity. Cell lines retained measurable TAT activity following long-term culture and frozen storage. TAT activity of transformed cells was comparable to that of normal lines. Prolonged frozen storage did not induce transformation up to one year.     

In vitro evaluation of some latent radioprotective compounds.

In tissue culture, protection against X-irradiation by a number of cysteamine derivatives was studied and the results were compared with data obtained in mice. Compounds with a covered SH group, like WR 638, cysteamine phosphate, WR 2721, and AE 48527, showed practically no protection when dissolved in tissue-culture medium, but developed a protective activity when dissolved in rat blood. Thiol measurements demonstrated that in rat blood the compounds were partly hydrolysed to thiols. C511 was also hydrolysed in culture medium and was slightly less effective than cysteamine in culture medium. Cysteamine phosphate was hydrolsed more easily than cysteamine sulphate and the protective activity in rat blood was better. WR 2721 was also partly hydrolysed in rat blood. The in vitro protection of this compound was disappointing when compared with results in vivo. Its SH form (WR 1065) also showed less protection than expected from in vivo experiments. Thus, the little protection by WR 2721 in vitro in rat blood is not only due to its incomplete conversion into its thiol. Longer incubation times and the use of rat blood as a solvent brought the protective activity of WR 1065 almost up to the level of cysteamine. This may indicate that WR 1065 penetrates the cells poorly. WR 1065 was the only compound we studied whose protective activity in vitro was improved appreciably by dissolving it in rat plasma.