DMPS (2,3-Dimercaptopropane-1-sulfonate, Dimaval) Decreases the Body Burden of Mercury in Humans Exposed to Mercurous Chloride 1

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1 /98/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 287, No. 1 Copyright 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 287:8 12, 1998 DMPS (2,3-Dimercaptopropane-1-sulfonate, Dimaval) Decreases the Body Burden of Mercury in Humans Exposed to Mercurous Chloride 1 DIEGO GONZALEZ-RAMIREZ, MIGUEL ZUNIGA-CHARLES, ANTONIO NARRO-JUAREZ, YOLANDA MOLINA-RECIO, KATHERINE M. HURLBUT, RICHARD C. DART and H. VASKEN APOSHIAN Department of Pharmacology, (D.G-R, M.Z-C, A.N-J, Y.M-R), Centro de Investigacion Biomedica del Noreste, Instituto Mexicano del Seguro Social, Monterrey, Mexico; Rocky Mountain Poison Center (K.M.H., R.C.D.), Denver, Colorado and Department of Molecular and Cellular Biology (H.V.A.), University of Arizona, Tucson, Arizona Accepted for publication April 20, 1998 This paper is available online at ABSTRACT DMPS (2,3-dimercaptopropane-1-sulfonate, Na salt), when used as a challenge test for mercury in workers involved in the production of a calomel skin-bleaching lotion and in direct contact with mercurous chloride, elevated urine levels of mercury. A DMPS treatment regimen was devised and initiated. Three days after the challenge test, DMPS was administered p.o. (400 mg per day) for 8 days, followed by a no-treatment period of five days. A new cycle of DMPS treatment for 7 days was initiated and followed by 5 days without treatment. A third period of treatment was begun for 6 days, followed by a 5-day Received for publication September 10, This work was supported in part by The Wallace Genetic Foundation, by the NIEHS Center grant P30ES06694 and by CONACYT grant 2167-M9303. no-treatment period. The urinary mercury greatly increased during those periods when DMPS was administered (1754, 314, and 173 g/24 h for the periods 1, 2 and 3, compared with 106, 48 and 53 g/24 h on the corresponding no-treatment periods). One of the workers presented signs of drug intolerance and was discharged after receiving the first cycle of treatment. DMPS treatment was effective in lowering the body burden of mercury and in decreasing the urinary mercury concentration to normal levels. The toxic effects of Hg 0,Hg and Me-Hg have been well investigated (for review see Clarkson et al., 1988), but those of Hg 2 have received very little recent attention. Mercurous mercury is the univalent state of mercury. Its compounds are binuclear and contain a mercury-mercury bond. Mercurous ions do not form many stable complexes. In the 1940s, teething powders for infants contained calomel (mercurous chloride) because of its antibacterial activity. Its addition caused acrodynia, also called pink disease, in some children (Warkany and Hubbard, 1951). Calomel is no longer used for such purposes (cf. Warkany, 1966). Mercurous salts have been used also for other medicinal purposes. For example, many Chinese patent medicines used in China and the Western world contain mercurous chloride (Kang-Yum and Oransky, 1992). Mercury has also been used as an antiseptic, a diuretic and an abortifacient. (Goldwater, 1972). In humans, DMPS (Dimaval) is an effective mobilizing agent for mercury (for reviews see Aposhian et al., 1995 and Aaseth et al., 1995). Compared with previously used antidotes such as BAL (Dimercaprol), it has many advantages, such as less toxicity and the availability of both oral and parenteral preparations (Aposhian, 1983; Aposhian et al., 1995). More is known about the pharmacokinetics of DMPS, given p.o. or i.v., in the human than about any other dimercapto chelating agent (Maiorino et al., 1991; Hurlbut et al., 1994; Aposhian et al., 1995). We have reported previously that workers exposed to mercurous chloride during the manufacture of a skin-lightening lotion, users of the lotion and non-exposed controls were given DMPS as a challenge test to evaluate the body/kidney burden of mercury (Maiorino et al., 1996). All the subjects responded to the challenge dose of DMPS by increased urinary excretion of mercury. Mean urinary mercury for the workers before the DMPS administration was 333 g/l, with a range of 57.7 to 1077 g/l. After the DMPS challenge, the mean urinary mercury was 4282 g/l 479 S.E.M., with a range of 2051 g/l to 7956 g/l (Maiorino et al., 1996). This paper reports the results of a second challenge test given approximately 18 months later and of a DMPS treatment regimen designed to increase the urinary excretion and decrease the body burdens of mobilizable mercury. The physicians waited 18 months to perform another challenge test ABBREVIATIONS: DMPS, sodium 2,3-dimercaptopropane-1-sulfonate; BAL, British Anti-Lewisite; Hg 0, elemental mercury; Hg 2, mercurous ion; Hg, mercuric ion. 8

2 1998 DMPS and Mercurous Hg 9 and treatment because the workers took that amount of time to decide whether they wanted to be treated. Materials and Methods The present study was approved by the Bioethics Committee of Instituto Mexicano del Seguro Social. Informed consent was obtained from each of the participants. The Dimaval capsules (100 mg of DMPS each capsule) were a gift of Heyl, Berlin. Dimaval is an investigational drug in the United States, so the study was performed under the U.S. Food and Drug Administration IND N o. 34,682. Because 18 months had elapsed since the first challenge test, a second test was performed before the initiation of the treatment. The second challenge test used a protocol identical to the previous one (Maiorino et al., 1996). The DMPS challenge and treatment were performed in Monterrey, N.L., Mexico, a city 400 miles away from the factory in which the participants had been exposed to mercurous chloride. This was done to be certain that exposure was not continuing during DMPS treatment and in order for supervisory medical personnel to be in attendance. Blood chemistry data were collected for each patient at eight time-points that spanned a 1-month period. Blood chemistry testing included 35 specific parameters: glucose, urea, BUN, creatinine, uric acid, direct bilirubin, indirect bilirubin, total bilirubin, cholesterol, total protein, albumin, globulin, albumin to globulin ratio, calcium, phosphate, alkaline phosphatase, lactate dehydrogenase, (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), RBC count, hemoglobin, hematocrit, mean corpuscular hemoglobin concentration, leukocytes, lymphocytes, monocytes, basophils, eosinophils, neutrophils, myelocytes, metamyelocytes, nuclear bands, segments, platelets and gamma glutamyl transferase (GGT). Hematologic studies were performed before and after the administration of the challenge test and before and after each DMPS treatment period. Treatment regimen. Periods of DMPS treatment and periods of no treatment were used in order to ensure that the increased urinary excretion of mercury was due to DMPS (table 1). The first cycle of DMPS treatment was started 3 days after the challenge test and consisted of the p.o. administration of 400 mg of DMPS per day as three divided doses for 8 days. The doses consisted of 100 mg before breakfast, 100 mg before lunch and 200 mg before dinner and were given 1 h before each meal. This first cycle of treatment was followed by 5 days of no treatment. On predetermined days of both the treatment and the no-treatment periods, 24-h urines were collected and the mercury levels measured. After the first period of treatment and no treatment, a second cycle of 7 days of treatment with DMPS at the same dose was initiated, followed by another no-treatment period of 5 days. At the end of this last period, a third cycle of 6 days of DMPS treatment was planned. This treatment sequence was to be continued until a normal range of urine mercury levels was obtained (below 50 g/g creatinine; WHO, 1991). The DMPS dose was chosen on the basis of previous studies using DMPS at the University of Arizona (Maiorino et al., 1991; Aposhian et al., 1992b) and those of Campbell et al. (1986), dealing with the treatment of elemental mercury poisoning. TABLE 1 DMPS Treatment Protocol Dimaval was administered p.o. 1 h before breakfast, lunch and supper at doses of 100, 100 and 200 mg, respectively. Days Treatment 0to3 no 4 to 11 DMPS(400mg/day) 12 to 16 no 17 to 23 DMPS(400mg/day) 24 to 28 no 29 to 34 DMPS(400mg/day) 35 to 38 no Urinary mercury analysis. Urine was collected in acid-washed 3-liter polyethylene collectors (Baxter). Concentrated HCl for trace metal analysis (Baker analyzed; 38.0%) was added immediately to give a final concentration of 1.8%. The acidified urine was transferred immediately to polyethylene bottles and frozen. All glassware or plasticware was soaked in 2% nitric acid at least overnight or washed with 25% nitric acid. All urine samples were acid-digested, at least in duplicate. Total mercury in each digested urine sample was quantitated by three cold-vapor generation/atomic absorption spectrophotometric determinations as described previously (Aposhian et al., 1992a; Gonzalez-Ramirez et al., 1995). Mercury content of urines was analyzed at the Centro de Investigacion Biomedica del Noreste Research Laboratory of the Instituto Mexicano del Seguro Social in Monterrey, N.L., Mexico. Validation of mercury assay was as follows: Mercury nitrate was added to freshly voided urine from a normal individual to give final concentrations of 0.50, 5.0 and 30 ng/ml of mercury. Six determinations of the 0.5 ng Hg/ml concentration gave a mean of 0.40 ng/ml (range, ), six determinations of the 5 ng/ml concentration gave a mean of 4.9 ng/ml (range, ) and six determinations of the 30 ng/ml concentration gave a mean of 28.9 ng/ml (range, ). Data analysis. Statistical analyses were performed using commercial statistical software, Graphpad InStat version 2.05a. Statistical analyses consisted of repeated-measures analysis of variance, ordinary one-way analysis of variance and nonparametric Kruskal- Wallis testing. A P value less than.05 for these tests warranted further analysis with Dunnett s Multiple Comparison Post-Test. Results Study population. Eight workers (five males, three females), aged 21 to 57 years, who had been exposed occupationally to mercurous chloride for 2 to 15 years participated in this investigation. They were employed in a factory located in Tampico, Mexico, that compounded and packaged a skin lotion used for cosmetic purposes. They had responded to the first DMPS-Hg challenge test with a very high level of urinary mercury. Clinical. Physical examinations performed before DMPS treatment indicated that the subjects appeared to be in acceptable health except for modest alterations in mood (anxiety, insomnia and occasional headaches) and other complaints mentioned below. One of the subjects was hypertensive and under treatment with a -blocker compound (propranolol, 120 mg/day), which treatment continued during the study with no obvious alterations in health or arterial pressure. Three subjects had 2 to 14 amalgam dental restorations. Before treatment, one of the participants complained of sporadic episodes of paresthesis in lower limbs; another worker had suffered from episodes of headache treated with common over-the-counter analgesics. In spite of the high levels of urinary mercury found, the mean urine protein was 45 mg/l (range mg/l). Normal levels are 10 to 200 mg/day (Med. Inf. Syst. 1996). Most of the blood chemistry parameters had individual values outside the normal range, but they were not sustained nor were they considered clinically significant. However, two of the blood parameters found before DMPS administration indicated abnormal values that may be due directly to mercury. The mean values for RBC counts and hemoglobin levels increased during the study period. This may be due to DMPS treatment decreasing the total body burden, because mercury exposure can depress red cells and hemoglobin levels (Woods et al., 1990). However, with such a small patient population, it is difficult to gauge the significance of this.

3 10 Gonzalez-Ramirez et al. Vol. 287 During DMPS treatment, one patient presented scarce, scattered maculo-papular 2 to 3-mm itchy lesions on both forearms. She reported gastric irritation and presented elevated AST, ALT and gamma glutamyl transferase (GGT) levels (220 I.U./l, 182 I.U./l and 423 I.U./l, respectively) in the blood samples taken at the end of her first course of DMPS administration. Normal ranges are from 9 to 40 I.U. (AST), from 13 to 48 I.U. (ALT) and from 4 to 18 I.U. (GGT) for the laboratory that did the exams. These adverse events were attributed partially to the concomitant consumption of alcohol during DMPS treatment. She elected not to continue in the study after the end of the first course of treatment. Ten days later, her physical exams and laboratory tests were in the normal range except for her GGT (AST, 43 I.U.; ALT, 46 I.U.; GGT, 346 I.U.). For the other subjects, the only complaints were of bad taste and nausea at the beginning of the first course of treatment. Two of the participants also reported headache at the beginning of the first course of DMPS. Urinary mercury excretion. The amounts of mercury mobilized and excreted in the urine by the second DMPS challenge (fig. 1) were very similar to those obtained in the first challenge test performed on the same subjects 18 months earlier (Maiorino et al., 1996). The second DMPS challenge resulted in a 44-fold increase in the excretion of urinary total mercury. The very high levels of urinary mercury after the DMPS challenge suggested that these eight subjects might benefit from sustained DMPS treatment to decrease the body burden. The therapeutic protocol was initiated 3 days after the second challenge test. The amounts of mercury eliminated during the first course of DMPS treatment (table 2; fig. 2) were not small in magnitude. The range of urinary Hg eliminated during the first day of the first DMPS treatment was 1441 g to 7269 g. The amounts and concentrations of urinary mercury for the seven patients who received a second and a third course of DMPS treatment decreased progressively (figs. 2 and 3; table 2). Discussion Fig. 1. Urinary mercury excreted before and after DMPS during the first (left; n 11) and second (right; n 8) DMPS challenge tests. s of urinary mercury were 16 to 314 g and 1728 to 10,307 g (before and after DMPS respectively) for the first challenge test and 28 to 399 g and 1393 to 7826 g for the second challenge test. Eighteen months elapsed between the two challenge tests. The occupational exposure of the factory workers in this study was to calomel, which they added as the putative active ingredient during the large-scale formulation of a skinbleaching cosmetic lotion. The concentration of calomel in this lotion has been reported to be 6 to 8% (Villanacci et al., 1996). Calomel is very insoluble. We do not know whether the workers inhaled mercurous chloride dust or absorbed it topically during their processing of large volumes of aqueous suspensions. Observations of the workers performance suggest that exposures occurred by both routes. Most toxicologic knowledge of mercury is based on studies of mercurials (Hg 0,Hg ) other than mercurous compounds. Knowledge of mercurous mercury toxicokinetics is very limited. In those reports where absorption, after very high exposure to mercurous mercury, could be proved, necropsial data indicated the presence of mercuric ions in tissues (Clarkson et al., 1988). The results obtained with the two challenge tests, performed 18 months apart, were very similar, although working conditions in the factory were improved after the first challenge test. We do not know whether this similarity was due to continued exposure or whether the second challenge test mobilized Hg that was more tightly bound in the tissue. The urinary mercury levels resulting from the first day of DMPS administration in each treatment course can be regarded as a measure of the magnitude of the mobilizable body burden of mercury at that time (figs. 2 and 3). There was a progressive lowering of urinary mercury after the first day of each DMPS treatment, which indicated that the increased urinary Hg excretion was the result of DMPS administration (figs. 2 and 3). Dental amalgams were present in three of the participants. The amalgams did not appear to influence the results of the present study, because their urinary mercury was much greater than that of the 13 control subjects on a previous study (Gonzalez-Ramirez et al., 1995) who were not exposed to mercury in the workplace but did have dental amalgams. In the previous study, their urinary mercury level before DMPS was 3 g/l and 37 g/l after the DMPS-Hg challenge test. The World Health Organization has recommended a maximum individual urine mercury concentration of 50 g/g creatinine (WHO, 1991). Those subjects who completed the three courses of DMPS had at the time of discharge 36 g Hg/g of creatinine, were excreting 53 g of Hg/24 h and appeared to be in excellent physical condition. They returned to work in the factory. Additional protective measures were recommended to, and adopted by, the employer to decrease further exposure to mercurous chloride. Since then, however, the factory has been closed to stop distribution of the lotion (Villanacci et al., 1996). A report on deleterious effects of this skin-bleaching lotion has appeared recently (Villanacci et al., 1996). The mean urinary mercury concentrations reported by Villanacci et al. (1996) in three mercury lotion users (143 and 355 g/g creatinine and 178 g/l were apparently higher than those found in users of the same lotion in a previous study (range 3.1 g/l 186 g/l; mean 63.5 g/l; Maiorino et al., 1996) but are lower than those found in the makers of the calomel skin lotion who are the subjects of this report. The Mexico Department of Health confiscated 35,000 bottles of this calomel lotion produced at the factory to prevent any further exposure to consumers (Lombera-Gonzalez et al., 1996). Use of this lotion has not been restricted to just a few people. Reports of toxic effects caused by its use probably will

4 1998 DMPS and Mercurous Hg 11 TABLE 2 Urinary mercury excreted during periods of DMPS treatment and periods of no treatment The mean of total mercury per person in 24 h and of concentration figures ( g/l and g/g of creatinine) are given. In parentheses are the number of days in which 24-h urine samples for all subjects were analyzed in each period. Cr creatinine. All subjects (n 8) were treated during the DMPS treatment period. One subject elected to be eliminated from the DMPS 2 and DMPS 3 periods. Regimen Days on Regimen g/24 h g/24 h g/l g/l g/g Cr g/g Cr No treatment 3 (2) DMPS 1 8 (6) No treatment 5 (3) DMPS 2 7 (5) No treatment 5 (3) DMPS 3 6 (4) No treatment 3 (2) Fig. 2. Decrease of the body burden of mercury by DMPS as indicated by mean 24-h urinary mercury excretion. DMPS (400 mg per day in three divided doses) was given on days 4 to 11, 17 to 23 and 29 to 34. Error bars indicate S.E.M. Fig. 3. Urinary mercury concentrations with and without DMPS treatment (400 mg per day in three divided doses was given on days 4 to 11, 17 to 23 and 29 to 34). Error bars indicate S.E.M. increase. A survey has been instituted by the Mexico Department of Health. One factor that may be involved in some of the severe effects seen after exposure to inorganic mercury is a possible immunological component (Pietsch et al., 1989). This factor may have been implicated in the acrodynia observed in children exposed to mercurous mercury as a component of teething powders. Only a few of the exposed children had acrodynia, irrespective of the calomel dose (Magos, 1975; Gotelli et al., 1985; Goyer, 1996). Whether the cases reported by Villanacci et al. (1996) are due to a similar atopic response is unknown at present. Mercury has been known since ancient times; it has been used as a medicine, as a poison and as a cosmetic. In more recent times, BAL has been used as an antidote (Klaassen, 1996). With the extensive use of DMPS, especially in Germany (Aposhian, 1983; Schiele et al., 1989; Kemper et al., 1990) and the former Soviet Union (Klimova, 1958), its use as a mercury antidote is encouraging. Acknowledgments The authors express their appreciation to Dr. Salvador Said Fernandez (Director of the Centro de Investigacion Biomedica del Noreste, Instituto Mexicano del Seguro Social, Monterrey, N.L., Mexico) for his encouragement, helpfulness, cooperation and support. References Aaseth J, Jacobsen D, Andersen O and Wickstrom E (1995) Treatment of mercury and lead poisoning with dimercaptosuccinic acid and sodium dimercaptopropanesulfonate: A review. Analyst 120: Aposhian HV (1983) DMSA and DMPS: Water-soluble antidotes for heavy metal poisoning. Annu Rev Pharmacol Toxicol 23: Aposhian HV, Bruce DC, Alter W, Dart RC, Hurlbut KM and Aposhian MM (1992b) Urinary mercury after administration of 2,3-dimercaptopropane-1-sulfonic acid: Correlation with dental amalgam score. 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