Pharmacology and toxicology of heavy metals: Mercury

Pharmac. Ther.A. Vol. 1, pp.

131-151, 1976, Pergamon Press. Printed in Great Britain

Specialist Subject Editor:

W. G . L E V I N E

PHARMACOLOGY AND TOXICOLOGY HEAVY METALS: MERCURY

OF

HAROLD G. PETERING and LLOYD B. TEPPER Department of Environmental Health, University of Cincinnatti College of Medicine, Cincinnatti, Ohio, USA

1. BACKGROUND Mercury has been known for thousands of years. Cinnabar (HgS) in the most widely mined form of mercury, and was the source of the metal as early as 1100 Bc in China. Mercury was first known in the Western hemisphere in Peru about 500 ac. Although mercury is widely distributed in the earth's crust, it is only found in traces in most rock formations. It was very early demonstrated in seawater by Proust (1799), and later shown by Stock and Cucuel (1934) to be present in the North Sea, in fresh waters, and in rainwater. Aydin'yan (1962) found mercury in many rivers in Eastern Europe, in the Black Sea, the Mediterranean Sea, the Atlantic Ocean, and the Indian Ocean; the amounts in all cases were in the range of 0.09 to 2.8 ppb. The U.S. Geological Survey (1970) recently has reported that the level of mercury in fresh waters of the United States was less than 3 ppb. There is very little information about the amount and distribution of mercury in the atmosphere, but it is assumed to exist there, since many mercury compounds and the metal itself are volatile, and its presence in rain water suggests that it is being washed out of the air. Williston (1968) found that mercury was present in the range of 1-10/~g/m 3 in the air over Palo Alto, California, with peaks up to 50/~g/m 3. At 10,000 ft altitude 20 miles wes.t of Palo Alto over the Pacific Ocean, the levels were only 0.6--0.7/~g/m 3. Recently McCarthy and co-workers (1969) of the U.S. Geological Survey reported that mercury in the air 400 ft above ground in the south-western part of the United States in a non-mineralized area was 3-9/~g/m 3, while above rocks rich in mercury the amount could reach 20/~g/m 3. Since mercury is apparently ubiquitous, we must expect to lind it in plants and animals. It is concentrated by some plants, by plankton, and by fish, and is absorbed through the skin (Warren et/al., 1966; Zimmerman and Crocker, 1944; Gibson et/al., 1941). Recently it has been found in fish and sea foods, in amounts which have alarmed many of the western countries. Its presence in considerable amounts in other foods, such as grains and vegetables, is probably the result of the use of mercurials as seed disinfectants and fungicides. Man has used mercury in increasing amounts in the manufacture of electrical equipment, scientific instruments, explosives, chemicals, and in the electrolytic production of chlorine and alkali (Stokinger, 1963). The metal is found in coal and petroleum, and during combustion much of it enters the general environment through stack emissions. In addition to direct occupational exposure, man is thus indirectly exposed to mercury found in industrial waste products. Many of these waste products are disposed of in rivers, lakes, and land falls, so that there is the possibility of rather widespread exposure of the general public to mercury. It can be estimated that the amount probably released into the general environment of the United States in the past half century is about 160,000,000 lb. Furthermore, mercury has recently become an environmental hazard because of its use as seed disinfectants in agriculture and as antifungal agents and slimicides in agricultural and paper pulp operations. Mercury compounds have long been used by physicians. Though many of these medical uses are on the wane, a number of mercurials are still employed as diuretics and as antiseptics. The dental profession also uses a large amount of mercury in silver 131

132

HAROLDG. PETERINGand LLOYDB. TEPPER

amalgams for dental fillings. Mercury compounds have also been used as antisyphilitics and cathartics for many years. Some are still listed in official codices and pharmacopoeias, but only the mercurial diuretics have retained a place in medical practice today, being of value in the management of cardiac edema. A list of mercurials which are presently available include the following: (a) Bichloride of mercury, mercuric chloride, HgC12. Used as an antiseptic for inanimate objects and unabraded skin. (b) Ammoniated mercury, HgNH2CI. Used topically in white ointment as an antiseptic. (c) Phenylmercuric nitrate, or basic phenylmercuric nitrate (mixture of phenylmercuric nitrate and phenylmercuric hydroxide). Used topically as an antiseptic and antifungal, and is a potent spermicide. (d) Thimerosal-Merthiolate ® Sodium Ethylmercury-thiosalicylate.

COONa ~

S"--HgC2H~

Antiseptic. Relatively non-toxic antibacterial agent. Used to disinfect skin surfaces. (e) Meralluride-Mercuhydrin®, N-{[3-(hydroxymercuri-2-methoxypropyl)]carbamoyl} succinamic acid plus theophylline (as sodium salt), theophylline---Hg--CH2---C H---C H2--N---C O---N--C O(CH2)---COOH

I

OCH3

H

H

Diuretic: Used as parenteral solution. (f) Mercurophylline, Mercuzan ®, Mercurin®, Mercuzanthin ®, N-(y-hydroxymercuri-/3-methoxy)propyl camphoramic acid. (As sodium salt.)

HOOC~

CO---NH--CH2--CH--CH2--HgOH

I

OCH~ Diuretic. Used as a parenteral solution. (g) Mercaptomerin-Thiomerin®, Sodium salt of N-(3,-carboxymethylmercaptomercuri-/3-methoxy)propyl camphoramic acid, cf. Mercurophylline

HOOCh]__ CO---NH---CH~--~H--CHz--Hg--S--CH2---COOH OCH~ Diuretic. Used parenterally. (h) Mersalyl-Salyrgan® (with theophylline), Sodium-O-{[3-hydroxymercuri-2methoxypropyl]carbamoyl} phenoxyacetate. Sodium hydroxymercurimethoxypropyl carbamoylphenoxyacetate

c.

..o.

OCH3 Diuretic. Used parenterally. (i) Chlormerodrin. Neohydrin®. [3-(chloromercuri)-2-methoxypropyl]urea. Diuretic. Used orally; also with '97Hg label for diagnostic purposes in kidney and brain disease.

Mercury

133

2. CHEMISTRY 2.1. INORGANIC MERCURIALS

In the periodic table of elements, mercury (Hg) is the heaviest member of the IIB group, which also contains zinc (Zn) and cadmium (Cd). Mercury is the only metallic element existing in the liquid state at ordinary temperatures and pressures; it has an atomic number of 80 and at. wt 200.59. It occurs naturally in the form of seven stable isotopes, ~S*Hg, ~ H g , ~Z~Ig, 2°°Hg, 2°~Hg, ~ H g , 2°'Hg which range in abundance from 0.16-29.72 per cent. Its pharmacology and toxicology can only be adequately understood on the basis of the variety of chemical reactions which mercury and its ions can undergo. Whereas Zn and Cd have very similar chemical properties, the chemistry of Hg differs markedly from that of the other two elements of Group IIB. Mercury exists in two oxidation states, Hg + and Hg 2+. In the Hg + state it exists as the metal-metal ion pair +Hg-Hg ÷ or Hg22+, so that the halide salts are Hg2X2. All mercurous halide salts except the fluoride are very insoluble and not readily hydrolyzed. The difficulty of oxidizing Hg ÷ to Hg 2÷, together with the insolubility of HgzCI2, probably accounts for the lack of serious toxicity of mercurous chloride, calomel (HgzC12). Although Hg + salts are not readily oxidized to Hg 2+ salts, the mercurous ion is stable to disproportionation by only a small margin, and therefore any anion which will reduce the activity of Hg 2+ ion will favor disproportionation as indicated in the following equation: Hga2÷ ,

, Hg ° + Hge÷AG = 3 kcal, K(Hg2÷/Hg2 2) = 6.0 x 10-'.

Similarly, Hg22++ 2OH-

Hg ° + HgO + H,O.

This disproportionation reaction has a very low redox potential so that when Hg ° and Hg 2+ are together only Hg22+ will result. Hg 2+ forms much more stable complexes with most ligands than does Hg2z+. As a result, Hg22+ disproportionates, yielding the complexes of Hg 2÷ and metallic mercury, and hence very few mercurous complexes are known. Mercuric oxide is relatively soluble in water, yielding solutions in the range of 10-3 to 10-'M, depending on the temperature, but the sulfide is extremely insoluble, having a solubility product of 10-s'. All four mercuric halides are known. The chloride is the most important one for our consideration, since it has been used as a disinfectant and in commerce. HgCI2 has considerable covalent character and is readily soluble in water, ethanol, and other organic solvents. Its solubility in water is 0.48 mol per 100 tool of solution (0.27 M) at 25°C, while the solubility in ethanol and ethyl acetate at 25°C is 8.14 and 9.42 mol per 100 mol of solution, respectively. The great lipid solubility of HgCI2 probably is an important factor in its toxicity because it permits penetration of membranes and entry into tissue rich in lipid, such as the central nervous system. Ammoniated mercury (HgNHeC1) was used widely in the past as an antiseptic in ointment form. It is obtained by reacting HgC12 with dilute ammonia so that no excess of NH4CI is formed. In the presence of excess NH4C1, Hg(NH3)2CI2 is formed which has no therapeutic use. The antiseptic quality of HgNH2CI probably stems from slow release of Hg 2+. Its insolubility and lack of ionization are probably due to the polymeric structure of the compound. Other significant chemical properties of inorganic mercury compounds are their reactions with halide anions, especially chloride ion, and with nitrogen and sulfur ligands. Sneed and Brasted (1955) and Webb (1966a) have reviewed in detail the reaction of Hg 2+ with C1-, and give the dissociation and ionization constants of the many products. The K,~,o~ of HgCI2, which is defined below, is 6.03 x 10-1" (i.e. Kt x K2). Thus pK~ is 6.74 and pK2 is 6.48, which means/3 is 13.22 and in an aqueous solution of HgC12 there is very little Hg 2+.

134

HAROLD G. PETERING and LLOYD B. TEPPER

K, = (HgCl-) x (Cl-)/(HgCl2) = 3.31 × l0 -7 K2 = (Hg 2*) × (CI-)/(HgCI-)

KtK2 = Kd,...... /3 = 6.02 x 10-4 = 1.82 × l0 -~. However, if HgCI, were present in biological systems, CF would be present in physiological amounts, 0.13-0.15 M; in sea water the chloride ion would be higher. In the presence of Cl-, HgCl~ and HgCI7 are formed. Sillen (1949) has shown that at 0.126M CI, HgC12, HgCl~, and HgCl2 are present at equimolar quantities, and the species HgCl- and Hg2÷ exist only in minor amounts. Br- and I- have even greater effects on the ionization of mercurials since they bind Hg 2÷ more strongly than does C1-. Since the pH of most biological systems and physiological fluids is above 7.0, the reaction of mercurials with OH- must also be considered. The pKl for the dissociation of Hg(OH)2 is 10.3 and that for pK2 is 11.4; thus in a chloride-free medium there would be very little Hg2÷. In fact, Webb (1966a) has estimated that Hg(OH)2/Hg2÷would be 5 × 103 at pH 5.0 and 5 × 107 at pH 7.0. Since the affinity of Hg 2÷ for OH- is greater than that for Cl-, one might expect that at physiological pH most of the mercury would be in the form of Hg(OH)2. However, since in most physiological systems the pH would be about 7.4 and the Cl- concentration about 0.15 M, it may be seen that CI-/OH- would be about 106, and so C1- will compete effectively with OH- which allows a predominance of the forms HgCI(OH) and HgCI(OH)~ along with HgCI2. Reactions of Hg 2÷ with nitrogen and sulfur ligands are important in understanding the biological activity of organic mercurials; we shall delay our discussion of this aspect until later in the article. 2.2. ORGANIC M E R C U R I A L S

There are three types of organic mercurials which must be considered in any discussion of the pharmacology and toxicology of mercury. These are (1) alkyl mercurials which we shall designate as R2Hg or RHgX; (2) aryl mercurials, which we shall refer to as Ar~Hg or ArHgX; and (3) a special group of alkyl mercurials which constitute the mercurial diuretics. In the case of RHgX, the compounds are non-polar and lipid soluble if X is CI, Br, I, CN, or CNS, and the HgX bond is covalent. However, when X is SOz or NO~ the HgX bond is ionic and the compounds have much salt-like character and are water soluble. The dialkyl and diaryl mercurials are non-polar, volatile liquids or solids with low melting points. They have low reactivity toward oxygen, water, and organic functional groups; but in acid solutions the Hg-C bond is readily broken to yield RHgX or ArHgX (e.g. R~Hg + HX ~ RHgX + RH). If there is substitution in the aryl ring as in the case of p-mercuriphenylsulfonate ion, the polar character of the compound is enhanced. Hughes (1957) has suggested that the C-Hg bond in CHHg + is a stable covalent bond, that the bond in ArHg ÷ is less stable than in the methyl mercuric ion, but that both bonds resist cleavage in physiological processes. Heslop and Robinson (1967) point out that those organic mercurials which are stable to air and water are relatively reactive with other metals in their zero valence state. They also indicate that C-Hg bond is a weak one, having a strength of 15 kcal, and that the lack of reactivity of some of the organic mercurials as well as of metallic mercury with oxygen is due to the great weakness of the HgO bond. Webb (1966b) has summarized the differences between organic and inorganic mercurials in a manner which aids in understanding their contrasting biological activities. (1) HgCL forms linear bifunctional complexes (HgL2) while RHg ÷ forms mono-functional complexes of the general formula RHgL; (2) the organic mercurials are less soluble in aqueous media than are the inorganic mercurials; (3) the unsubstituted organic mercurials and their monofunctional complexes are much more soluble in lipids than are the mercuric salts or complexes; (4) although the affinity of RHg ÷ is less for many ligands than is that of Hg 2÷, the reactivity of both forms of mercurials with SH groups is greatly reduced by the presence of CI-, OH-, and amino acids or other sources of nitrogen ligands; and (5) the weakness of the C-Hg bond of

Mercury

135

organic mercurials is of importance in understanding the movement and distribution of Hg 2÷ in biological tissues when one of the former has been administered. The covalent nature of CH3HgX is shown, for example, in the dissociation of several forms of X, (Huges, 1957). The p K is 17.0 for RS-, 9.0 for I-, 7.0 for Br-, and 5.7 for CI-. These values show why alkyl mercurials are almost always found bound to sulfhydryl groups in biological tissue. The mercurial diuretics are a special class of alkyl mercurials, being primarily of the formula RCH2CH(OY)CH,HgX where Y is H or alkyl and X is usually OH or acetyl. It appears that the presence of the OY group adjacent to the HgX group increases the covalent nature of the HgX bond and reduces reactivity with tissue other than the kidney, R may be any number of groupings found by empirical testing to produce certain pharmacological properties. 2.3.

REACTION OF MERCURIALS WITH SULFHYDRYL GROUPS

Hg 2+ forms strong covalent bonds with RS-, yielding RSHg + and (RS)2Hg species, which have a linear or near-linear structure. Hg 2+ can also react with some di-sulfhydryl compounds such as dimercaprol (BAL) to form chelates such as CH~ CH2=~S~_ ~ i S / Ht~ CH2OH. This subject has been adequately reviewed by Webb (1966, pp. 746--751). The pKI for the reaction of Hg 2+ with cysteinate ion is 20.1 (Simpson, 1961), while the pI3(pK! +pK2) is 43.57 (Stricks and Kolthoff, 1953). By calculation the pK, is about 23.4. These data show that when two sulfur ligands are reactive in the same molecule, the second ligand binds as strongly as the first, which would suggest that wherever possible Hg 2+ would alter protein structure by forming R(SX)HG(SX)R complexes. Nevertheless, it can be shown that, in the presence of physiological concentrations of Cl- at pH 7.4, the binding of Hg 2+ to cysteine or glutathione will be considerably diminished. The amino acids, peptides, proteins, and NH3 or NI-I~ in tissues represent a pool of nitrogenous ligands with high affinity for Hg 2+. These, together with CF and OH-, constitute a group of ligands which will compete with RS- for Hg 2+ as well as for RHg + and ArHg +. It can be shown that the minimum lethal concentration of HgCl2 for bacteria can be increased six times by 67 mM concentrations of glycine, aspartate, glutamate, arginine, or lysine, and 120 times by the same concentration of cysteine (Webb, 1966c). Brooke and Davidson (1960), for example, give the pI32(pK1 + pK2) of Hg (histidine)2 as 21.2, and Webb (1966d) indicates that the value for amino acids other than cysteine lies in the range of 12-20. The p132 of mercuric complexes of purine and pyrimidine bases lies in the range of 11.5-21.2, and it is known that the phosphate and pyrophosphate groups have affinity for Hg in its various forms. Therefore, it is evident that the concentrations of Hg 2+ in any biological system will be extremely low. A similar picture can be developed for the organic mercurials, showing that the presence of the many mercury-binding ligands at physiological concentrations will greatly alter the formation of RHgSR'. Thus Hughes 0967) found that the binding of methyl mercuric ion with human serum albumin has a p K of 4.6, but Webb (1966e) calculated that the p K for binding to the sulfhydryl groups of serum albumin should be 22.0. The difference is ascribed to the action of competing ligands. From these and other data it may be inferred that either the reactive forms of Hg 2+ and RHg + are present in biological tissue at extremely low concentrations and thus are much more effective than the administered doses suggest, or that other species of mercury have pharmacologic and toxic action. It may also be seen that difunctional chelating agents such as dimercaprol and penicillamine will be much more effective in binding mercury for detoxification than will mono=functional ligands. Again it must be considered in any design of an agent for removing heavy metal whether the complexed form itself has activity. 3. ANALYTICAL DETERMINATION OF M E R C U R Y There are many acceptable methods for determining mercury, depending on the quantity to be analyzed and its chemical state in a given sample (Coetzee, 1961). The JPTA Vol. 1, No. 2=-B

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HAROLD G. PETERING and LLOYD B. TEPPER

most difficult task is the preparation of the sample because of the volatility of mercury and its compounds and the peculiarity of its chemistry, as discussed in Section 2. Attention must be paid to methods of extraction and ashing. This section will include a discussion as well as a critical evaluation or summary of the methods in use at this time. 3.1. PREPARATION OF SAMPLE

Inorganic mercury, such as that found in ointments, dusting powders, wood, preservatives, or plant sprays, which are H ~ + compounds (oxide, chloride, or amine chloride), can be extracted with 5% HCI. If Hg + as chloride is present, addition of a little bromine will convert this to Hg 2÷. A summary of methods for determining Hg in such preparations is given by Weissmann (1956). Some preparations of organic mercury can be decomposed to mercury metal directly by the use of SnCI2 in dioxane, or with hypophosphorous acid or ethanolamine in the presence of sodium. Usually, however, determination of mercury in organomercurials or in biological specimens requires preliminary decomposition of the organic matter by oxidation steps. The volatility of mercury metal and mercury compounds does not permit dry-ashing procedures, and even wet-ashing requires careful precautions to avoid losses of mercury during oxidation. Gorsuch (1970) has prepared a monograph on the choice of methods of destroying organic matter prior to analysis of inorganic constituents which should be consulted for details. Oxidizing agents, such as nitric acid alone or with sulfuric acid, nitric acid and H202 (Stock et al., 1933), nitric acid and permanganate (Winkler, 1935), have been used. Sulfuric acid with H202 (Parker et al., 1955), (Polley and Miller, 1955), ammonium persulfate (Stock et al., 1933) or KMnO4 (Booth et al., 1926) have also been used effectively. There is no agreement on the best method for this task but Stock and co-workers (1933) favored HCI-KC103 or HNO3-H202. The latter mixture decomposes biological samples more easily, but there may be losses during the digestion procedure. If HC1 is used, Cl- must be removed before the Volhard method for mercury is used, due to interference of chloride ion. Goldman and Jacob (1953) prepared urine for mercury analysis by refluxing the sample with HNOr-KMnO4. This has recently been shown by Kopp and Keenan (1963) to give very consistent results and recoveries of 100-+5 per cent. For very low concentrations of Hg the HNO3-KMnO4 digestion is followed by adsorption on anion exchange resin and elution with 0.002 thiourea-0.01 M HCI (Kopp and Keenan, 1963). With this method submicrogram quantities of Hg in 50-100ml of urine can be determined. 3.2. QUALITATIVE DETECTION OF Hg

Coetzee (1961) has summarized the important qualitative methods for mercury. Additional details are given in Feigl's classic text (1956). All mercury compounds give a gray to black deposit of Hg ° on being heated with Na2CO3 in a closed tube. The deposit forms mercury globules when rubbed with a glass rod. Hg ÷ is precipitated as Hg2CI2 in hydrochloric acid. In the dark it can be distinguished from ARCI by the addition of ammonia which causes the precipitate of Hg2CI2 to turn black but dissolves the ARCI precipitate. Hg 2÷ is most readily determined in HCI solution by passing in H2S. There is the formation of a white precipitate of HgC12.2HgS which rapidly turns yellow, then brown, and ends up being black HgS. HgS is insoluble in 8 M HNO3; this test eliminates other possible sulfides. Reducing agents such as SnCI2 will form Hg ° with either Hg + or Hg z+ compounds. This is the basis of present micromethods for mercury by atomic absorption spectrophotometer. Colorimetric tests can also be used to detect mercury. Thus, diphenylcarbazone reacts with either Hg + or Hg 2÷ in 0.2 M HNO3 to give a blue to violet color, which can be used to detect 1/.~g of mercury when present at a dilution of 20 ppm.

Mercury

137

Dithizone can also be used for Hg ÷ or Hg2+ to give yellow or orange complexes, respectively, which are soluble in CC14. The detection limit of dithizone is 0.25 #g at similar dilution limits.

3.3. QUANTITATIVE DETERMINATION Mercury has been estimated in many sources by gravimetric, titrimetric, polarographic, and photometric methods. Of all of these, the Volhard titrimetric method, the dithizone colorimetric, and atomic absorption spectrophotometric methods appear to be the most useful, consistent, and sensitive. (1) Volhard Titrimetric Method The Volhard titrimetric method involves titration of Hg 2÷ with SCN- using Fe 3÷ as indicator (Volhard, 1878). It is the recommended method providing the solution is free of chloride ions and other complexing substances. Fe 3÷ is usually present at 0.013 M. At the end point a faint pink color (FeSCN) 2÷ is seen which should be compared with standards. The titration accuracy, which is estimated at 0.1 per cent, may be improved by carrying out the titration at 15°C or below, or by introducing a potentiometric (amperometric) method of determining the endpoint (Kolthoff and Lingane, 1935). (2) Dithizone Colorimetric Method When organic sources of mercury are used, precaution must be taken to remove any excess of oxidizing solution in order to prevent destruction of the dithizone reagent. The solution to be analyzed may contain up to 0.5/zg of Hg ÷ per milliliter and mustbe about 0.5 M in H2SO, and 0.5 M in acetic acid. Twenty to twenty-five milliliters are mixed in a separatory funnel with 5 ml of chloroform and shaken well to saturate the aqueous phase with CHCI3. The organic layer is discarded and 5 ml of 0.001% dithizone in CHC13 is added and the mixture is shaken well for 1 min. The absorbancy of the CHC13 phase at 500 nm is determined and compared with a standard calibration curve. (3) Atomic Absorption Spectrophotometric Method This is the method of choice to determine Hg ° vapor. There are several variations which depend on different methods of preparing the analytical sample and of estimating the absorbancy. (a) Flameless atomic absorption photometry. Woodson (1939) developed a mercury vapor detector to determine Hg° in air which depended on the fact that mercury vapor in the path of light from a suitable mercury arc lamp giving the 253.7 nm resonance line would absorb this wavelength of light and the amount of absorbed light could be measured with a sensitive photoelectric cell. Ballard and Thornton (1941), recognizing the validity of the criticism of Hanson (1941) that some organic vapors could also absorb light at 253.7 nm presented a method by which Hg2÷ was removed from a solution as HgS and collected on an asbestos pad. This pad was placed in an apparatus in which the mercury could be vaporized and the vapors determined by absorption of 253.7 nm resonance line of Hg. The method allowed the determination of 0.06 - 0.02/~g Hg in 150-400 ml of solution. This method has gone through several modifications designed to improve the photometer (Zuehlke and Ballard, 1950; Ballard et al., 1954; Ling, 1967; Ling, 1968) and furnace (Vaughn and McCarthy, Jr., 1965). However, recent developments have made it possible to use conventional atomic absorption spectrophotometers or simple commercial instruments directly. Hatch and Ott (1968) showed that Hg 2÷ obtained from any source could be reduced with Sn 2÷ in acid media to give Hg°. When vaporized by aeration in the light path of a mercury hollow cathode lamp of an atomic absorption spectrophotometer, a sensitive measure was obtained of the mercury in the original sample. No flame was needed, and this method is known as 'flameless' atomic absorption. The method was sensitive to

138

HAROLD G. PETERING and LLOYD l]. TEPPER

100 ppb in solution, and recent developments suggest that the sensitivity can be further improved. Chau and Saitch (1970) using dithizone in chloroform as a means of concentrating Hg °, Hg ÷, and Hg 2÷ and of removing impurities before back extraction into hydrochloric acid showed that the 'flameless' atomic absorption photometric procedure could detect 0.008/~g/1 of solution, or 0.008 ppb. Goldberg and Clark (1970) found that by using mild oxidation of urine with H2SO4 and KMnO4 followed by extraction with dithizone in chloroform, 30-97 ppb Hg could be determined. Lindstedt (1970) found that a conventional U.V. absorption spectrophotometer could be modified so that 'flameless' atomic absorption (or cold vapor atomic absorption) could be used to detect 2/~g Hg/ml of urine if the sample had been previously oxidized at room temperature overnight with H2SO,-KMnO4 and then reduced with Sn 2*. (b) Flame atomic absorption photometry. Moffit and Kupel (1970) have presented a modification of the methods described above which permits the use of the conventional flame atomic absorption spectrophotometric configuration. They prepared their sample by suitable digestion, reduced Hg 2÷ to Hg ° with Sn 2÷ and vaporized it with a stream of air. However, instead of passing the vapors directly into an absorption cell in a spectrophotometer, they collected the vapors on a special form of charcoal. Thus they could collect and store many samples for future analysis. The charcoal adsorbent was analyzed for Hg by placing it in a tantalum boat which was then moved into the conventional flame of a Boling burner, and the absorption of light from a mercury hollow cathode lamp is measured in a conventional manner on a recorder or digital readout. With this technique they have been able to determine 0.02/zg per sample which is a sensitivity sufficiently high for many kinds of routine and research analyses. This latter procedure appears to have great merit if one has a conventional atomic absorption spectrophotometer available. Otherwise, the procedure of Lindstedt (1970) would appear to be the simplest method available for small amounts of mercury in biological samples. 4. BIOTRANSFORMATION OF M E R C U R Y The transformation of metallic, inorganic, and organic mercury in biological systems or by biologically controlled processes plays an important role in the ecological distribution of mercury. It determines the specific form of mercury to which man and animals may be exposed, and therefore the exact toxic and pharmacologic reactions which will follow exposure. Since biotransformation in man and animals begins as soon as there is intake of mercury or mercurials, this process has much to do with the absorption, distribution, organ localization, and excretion of the element or its compounds. The conversion of elemental mercury into the mono- and di-valent forms, as well as the disproportionation of Hg ÷ to Hg 2÷ and Hg ° were discussed above; these reactions also take place in biological systems. Both Hg 2÷ and RHg ÷ are capable of many reactions with anions and ligands which abound in biological and physiological media. The weakness of the C-Hg bond in organic mercurials permits the release of Hg 2÷ from organic mercurials, a process which may be involved in a mechanism of action of these organic compounds. Thus both mercurial diuretics and non-diuretic organomercurials release Hg 2÷ to animal tissues at similar rates after their administration (Clarkson et al., 1965; Clarkson and Greenwood, 1966). Furthermore, every class of organo-mercurials, including phenyl and alkyl mercurials, releases inorganic mercury in animal tissue (Miller et al., 1960; Miller et al., 1961; Gage, 1964; T. W. Clarkson, personal communication, 1971). Rothstein and Hayes (1964), have suggested that Hg ° vapor is oxidized to Hg 2÷ in the rat, and Clarkson (1965) has shown that this action occurs in vitro in the presence of heparinized blood. There is yet another type of biotransformation which occurs in nature and which may be important in understanding the toxicology and potential hazards of inorganic mercury. This is the biotransformation of inorganic mercury to organomercurials, primarily the formation of simple alkylmercury compounds, such as dimethylmercury

Mercury

139

or methylmercuric chloride. Wallace et al. (1971) present the following figure, which has been modified to describe the transformations of mercury and its compounds in nature: Hg ° ,

(CH,)2Hg

n

Hg °

Atmosphere

(CH3)2Hg

Biosphere

+-..... Hg 2+

(Agricultural and Industrial Fungicides)

, CH3Hg +

U

HgS (soil water) It demonstrates the importance of the interconversion of Hg ° to Hg2÷, and this in turn to (CH3)2Hg or CH3Hg+, and the manner in which all mercurials finally are a source of methylmercurials because all can dissociate to give Hg2+. The importance of these transformations is that we can no longer compartmentalize the study of the toxicity of mercury into elemental mercury, inorganic mercury, organomercury, or even therapeutic products or pesticides. Mercury, in any form and from whatever source, can be taken up by animals in the form of methyl mercurials which themselves can be formed from all other forms of mercury. In addition, as we shall see in a later section, we must now also consider the possibility of similar transformations in the intestinal tract of man and animals as well as during metabolic processes. The mechanism of the formation of methylmercurials (or alkylmercurials) has only recently been studied. Jensen and Jernel6v (1967; 1968) and Jernel6v (1969) reported that microorganisms could methylate inorganic mercury and suggested one of the following schemes as the probable route of reaction: Hg 2+

~Yt'~

Hg2+

m,~y~tt~ , (CH3)2Hg ~

ellzyme

~ CH3Hg+ (

, (CH3)2Hg

or

, CH3Hg+.

el~lzy~

In the first case, there would be a step-wise methylating with the intermediate methylmercuric ion being directly formed during the process; and in the second case, the primary product would be dimethyl-mercury with the formation of methylmercuric ion by dissociation, which occurs readily in acidic media. In 1968, Wood added another dimension to our understanding of this process when he found that the methylation of Hg2+ could take place non-enzymatically as well as enzymatically by a biological process involving methyl cobalamine (vitamin B,2). This process was originally postulated to be the following: Hg2+

, Hg(CH3)2 /

(CH3)2Hg(Stable) (CH3)Hg ÷ + CI-L

2CH3Bt2

2Bn

In a recent review of Wood's work in this area (1971), a number of possible mechanisms for the enzymatic and non-enzymatic formation of methylmercurials are discussed.

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Wood showed that dimethyl mercury was formed by an extract of a culture of Methanobacterium emilianskii in the presence of Hg 2÷, and that the same reaction would occur in solutions of methyl B12. Since the organism was a methane-producing one and since dimethyl mercury was formed, Wood reasoned that the reaction was taking place in a reducing environment. This led him to suggest that the enzymatic methylation may actually occur with Hg °, and the initial formation of the dimethyl derivative. Wood has shown that the methylation of Hg 2÷ by methyl corrinoids (compounds similar to vitamin B,2) is pH dependent and that the stoichiometry requires two mercuric ions to one molecule of methyl-corrinoid. He concluded that the non-enzymatic reaction does not occur in the presence of either Hg ° or mercurous ion (Hg22+), and that the enzymatic reaction would probably only occur in those aerobic organisms which use methyl corrinoids in their intermediary metabolism. West66 (1969) and Schwarzenbach and Schellenberg (1965) have shown that the very volatile dimethyl mercury, which is also lipid soluble, is sensitive to decomposition at acidic pH. It first forms the methylmercuric ion which then reacts with many anionic groups, including sulfhydryl, that occur in all biological tissue and fluids. West66 (1969), has shown that mercuric ion will be methylated in liver homogenates and that hens fed seeds treated with Hg 2÷, methoxymethylmercurials, or phenylmercurials, lay eggs which contain methylmercuric compounds in the white. Hammond (1971) has recently reported that mercury can be converted to methylmercurial by organisms residing in animal intestinal tracts. He has also suggested that the methylation of mercury can proceed more efficiently in aerobic than in anaerobic microbial systems. Norseth and Clarkson (1970) have found that methyl mercury ion is only slowly transformed to inorganic mercury, which is excreted in the feces and urine of rats. The transformation may be non-enzymatically catalyzed by the thiol groups of cysteine, glutathione, or proteins, a process that is especially marked in the liver. This biotransformation results in the localization of high levels of inorganic mercury in kidney tissue. 5. CLINICAL TOXICOLOGY OF MERCURY Clinical phenomena in man due to excessive exposure to mercury compounds reflect the route and intensity of exposure, and the chemical form of the metal. For practical purposes, toxic reactions due to inorganic as compared to organic mercury compounds can be considered to be different illnesses, with dissimilar symptoms and signs, pathogeneses, and responses to therapy. Intensity and duration of exposure in each case will influence the duration of clinical manifestations. 5.1. INORGANIC MERCURY POISONING

While the clinical literature does include multiple references to acute mercury poisoning, primarily from mercuric chloride usually in association with hospital accidents or homicidal or suicidal attempts, the more extensive historical accounts begin with occupational over-exposure in classical times. Pliney mentioned toxic effects among mercury miners in Almaden (Spain), and Paracelsus told of disease and exposure limiting 6-hr work days at Idrija (Yugoslavia) in the 15th century. The use of inorganic mercury compounds in the production of felt for hats led to chronic mercurialism among hatters in many countries throughout the 19th and early 20th centuries. In the United States, mercurialism among hatters became known as the Danbury shakes, since Danbury, Connecticut was the center of the hat trade during this period. In addition to mercury (cinnabar) mining and felt making, other trades in which mercury found use also contributed to the incidence of intoxication. These included mirror making, fire gilding, the production of thermometers, manometers, and barometers, chemical syntheses of mercury fulminate primers and mercurial fungicides, and a variety of operations in electrical and electronics industries. 5.2. CLINICAL MERCURY POISONING Acute exposure to soluble mercury compounds via the gastrointestinal tract is manifested in an intense irritating and necrotizing gastroenteritis accompanied by

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vomiting and diarrhea productive of blood and shreds of mucosa. Loss of fluids and shock may be overwhelming. Survivors of the immediate challenge may develop severe and fatal renal decompensation. In such patients hemodialysis has been life saving in that it preserves a reasonably normal nitrogen and potassium level in the blood while the kidney is recovering its normal function (Sanchez-Sicilia et al., 1963). Acute respiratory exposure to high concentrations of mercury vapors may lead to an erosive bronchiolitis and interstitial pneumonia which may be fatal (Matthes etal., 1958). In survivors of the acute episode dyspnea may persist for more than a year. Chronic mercurialism, due in most cases to the absorption of inorganic mercury via the respiratory tract, is the form of mercury intoxication most commonly observed in industrial situations (Kazantzis, 1965). The onset tends to be insidious and may be marked by a metallic taste, loss of teeth, and a gingivitis in which the gums are swollen and bleed easily. A metallic line on the gingival margin is rarely seen. The oral phenomena are exaggerated by poor oral hygiene. Illness may also be heralded by neuropsychological aberrations. The so-called 'erethism' of chronic mercury poisoning is characterized by an unusual self-consciousness, irritability, intolerance of criticism, and sudden changes in sociability. The patient may feel depressed and discouraged and may seek solitude. His attention and sleeping patterns may be disturbed. These changes are often first noted by the patient's family. Obvious neurological signs are seen in the tremors of chronic mercurialism. The fine static tremor first involves the fingers, eyelids, lips, and tongue and may progress to the arms and legs. This tremor m a y cause deterioration in handwriting and in the ability to perform other manipulative tasks. The gross tremors and 'shakes' described in historical accounts (Danbury shakes) are less commonly observed. Mercurialentis is a light to coffee brown discoloration of the anterior capsule of the lens (Kipling, 1965). The cause may be a prolonged local exposure to mercury vapors as may be experienced by an instrument maker. Corneal opacities may also occur. In some cases of chronic mercurialism, there is an increased excretion of albumin in the urine; casts may be seen. Renal failure, as seen in acute mercury poisoning, is not part of the clinical picture, however. The excretion of mercury in individuals with chronic mercury poisoning is highly variable, and is not of great help in defining the seriousness of absorption or of illness. In many situations, however, the excretion of 0.3mg of mercury over 24hr has been regarded as indicative of hazardous overexposure. Cases of clinical illness have been reported at levels less than this, and some persons who persistently have urinary mercury levels in excess of this show no disease. The treatment of chronic mercurialism is based upon the use of dimercaprol (BAL) or of penicillamine or its N-acetyl derivative (Smith and Miller, 1961). EDTA is contraindicated. Pink disease or acrodynia is a disease of infants and young children in which mercury has been implicated (Warkany and Hubbard, 1953). Acrodynia has been associated epidemiologically primarily with the use of body powders and talcs containing calomel. There is increased excretion of mercury, which has also been attributed, in various situations, to contact with calomel, ointments, bichloride of mercury diaper rinses, house paints, and unidentified sources of the metal. Clinical manifestations are dissimilar to those of mercury poisoning in the adult. The child is fretful, irritable, anorectic, and weak. There is photophobia, and cutaneous patches of erythema are seen. The hands and feet are red, swollen, and cold, and there may be inflammation of the mouth and gums. The pathogenesis of this disorder is not understood.

5.3. ORGANIC MERCURY POISONING Poisoning due to organic forms of mercury is primarily related to the absorption of alkyl mercury compounds. Although second degree skin burns may be caused by a variety of organomercurials, those in the phenyl and alkoxy-alkyl groups have produced no well documented cases of systemic intoxication. Disease due to alkyl mercury compounds was first associated with industrial exposure in the production of

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agricultural fungicides and seed dressings. Subsequently, similar manfestations were noted in non-occupational intoxications among persons using treated seed grain as a source of flour, and among persons eating fish which had taken up alkyl mercury compounds from contaminated bodies of water. Minamata disease (Kurland et al., 1960) is the syndrome which occurred in Japan during the years 1953-61, in which some 114 cases were reported with at least forty-four deaths. Congenital cerebral paresis was reported in nineteen infants, the mothers of whom often lacked symptoms. Epidemiological investigations related the disease to industrial inorganic mercurial effluents in a salt water bay, the biotransformation of these materials into alkyl mercury by bacterial and algal sediments in the water, and the investion of these organisms by food fishes consumed in large quantities by local fishermen and their families. A similar epidemic occurred in Niigata (Agano River) in 1965 (Tsuchiya, 1969). 5.4. CLINICAL INTOXICATION

The clinical pattern in alkyl mercury poisoning is reasonably consistent, whatever the source of metal. There is little if any salivation, stomatitis, or erethism as seen in inorganic mercury poisoning. Rather, the disease affects the nervous system, often with devastating consequences. Depending upon the intensity of exposure, the onset of toxic symptoms may be rapid, as in certain industrial situations, or it may follow a latent period of several month's duration. Early signs of toxicity are usually sensory disturbances with numbness and parasthesias. Motor impairment leads to ataxia, instability, dysarthria, dysphagia, and incontinence. The constriction of visual fields may be prominent with blurring and 'tunnel vision'. Central atrophy may be observed. Memory and intelligence are usually unimpaired. There may be partial recovery, especially when the dose has been small and the disease relatively mild. Permanent injury is common, however. Because of the advanced neurological impairment and disability, death from inanition and intercurrent disease may result. There is no effective therapy for organic mercury poisoning at the present time, and the prognosis in all but the mildest cases is not favorable.

6. T R E A T M E N T OF M E R C U R Y POISONING As was indicated in Section 5 above, the modern treatment of inorganic mercury poisoning in man is usually accomplished by the use of such chelating agents as 2,3-dimercaptopropanol (dimercaprol, BAL), oL-penicillamine or N-acetyl-DLpenicillamine (/3,/3-dimethylcysteine). This was introduced by Longscope and Luetscher (1946) and by Smith and Miller (1961). Other measures include the use of stomach lavage with sodium formaldehyde sulfoxylate to reduce mercuric ion to the less soluble mercurous ion. Moeschlin (1965) presents an excellent treatise on the treatment of acute and chronic mercury intoxication. In recent times it has become evident that alkyl and aryl mercurial poisoning warrent special attention, especially after chronic exposure. The use of BAL has come into question because of the work of Berlin and Ullberg (1963). They found that even though BAL increased the urinary excretion of mercury of mice given an injection of phenyl mercuric acetate and BAL (1 : 1) there was also an increase of mercury localizing in the brains of these mice in comparison with those receiving the mercurial alone. The possibility of forms of treatment other than BAL or penicillamine has been raised by the report of Selye (1970) that certain 'catatoxic steroids' especially spironolactone, prevent the nephrotoxic action of mercuric chloride in rats. Using 100 g female rats, Selye found that 100 #g of spironolactone given subcutaneously could prevent the nephrotoxic action of 400/~g of mercuric chloride given by stomach tube, Winter et al. (1968) found that glutamic acid in a number of molecular forms could prevent or alleviate the toxic action of mercuric chloride in rats. They showed that 2 g/kg of glutamic acid given orally had a pronounced preventive and curative effect against 10 mg/kg of mercuric chloride injected subcutaneously. Finally, Parizek and a

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group of Czech scientists (1967; 1968; 1969) have produced evidence that selenium compounds will protect against the toxicity of mercuric chloride. Their reports and those mentioned previously offer the possibility that there may be nutritional means of combating the toxic effects of environmental mercury as well as the chronic or acute condition found in workers of certain industries.

7. ABSORPTION, EXCRETION, AND DISTRIBUTION Since measure route of mercury

the clinical manifestations of mercury intoxication are dependent in large on the form of mercury to which an individual is exposed as well as on the ingestion, special consideration is given in this section to these aspects of toxicity. 7.1. ELEMENTAL MERCURY

Mercury vapor (Hg°) is present wherever mercury metal is used, whether in a dentist's office, a gas analysis laboratory, or a chloralkali factory. Elemental mercury is absorbed slowly through the skin but under most circumstances this is of little consequence. Oral absorption of metallic mercury is also not of great practical importance. Mercury vapor, on the other hand, is well absorbed from the respiratory tract, and exposure to the vapor presents a real hazard which must be controlled. The vapor pressure of mercury can be calculated from the formula in the temperature range of 0--1500C: log P = - 3212.5/T + 8.025 (Stokinger, 1963) It is such that at room temperature the concentration of mercury can reach 10 mg/m 3, which is 100 times the permissible industrial level (maximum allowable concentration, MAC). Although there is an epidemiological association between urinary excretion of mercury and exposure to mercury, the individual relationships are often inconsistent (Goldwater et al., 1962; Ladd et al., 1963). Nevertheless, Rentos and Seligman (1968), in a study of the exposure to mercury vapor and dust of eighty-three employees in nine mines and mills, found a very good correlation between urinary mercury and the presence of symptoms. They were also able to state that no worker with clinical symptoms of mercurialism has urinary mercury less than 300 p4g/l, which they consider the upper level of 'normal' values. Wada et al. (1969), studied forty-seven workers exposed to mercury vapor without clinical symptoms and concluded that the maximum permissible urinary concentration should be 200/zg/g creatinine. They also found that a decrease of 8-aminolevulinic acid-dehydratase and choline esterase of erythrocytes became prominent when urinary mercury rose above 200 p.g/g creatinine. Berlin et al. (1969a) have shown that mercury vapor penetrates the alveoli of guinea pigs exposed to either 0.1/~g or 5.0 mg/m 3 for 10 min and for 1 hr. Most of the mercury absorbed is quickly transferred to the blood, but 25-33 per cent remains in the lung and is only slowly eliminated. Berlin and co-workers (1969b) also showed that inhaled mercury vapor gets to the brain of rabbits and monkeys in ten-fold higher concentration than does injected mercuric ion. They suggest that there is no need to invoke the formation of organic mercurials from the vapor, since the lipid solubility and lack of charge facilitates Hg ° entry into tissue. In fact, animals exposed to Hg ° vapor have much more mercury in RBC than do those injected with Hg 2÷. In a third paper by Berlin's group (1966c), inhaled Hg ° was shown to cause greater deposition in the myocardium of mice than did HgCI2. They also found that acute symptoms of Hg vapor exposure were CNS and respiratory, but with injection of HgC12 acute symptoms were predominantly renal. Hg vapor is rapidly taken up by red blood cells and oxidized to Hg 2+, which is then equilibrated with plasma (Clarkson et al., 1961). After inhalation of vapor, blood Hg rapidly equilibrates with other tissues, including the brain (Magos, 1967). This author also suggested that the

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circulation time from lung to the brain of mercury vapor was very much more than could be accounted for by the slow oxidation of Hg°-* Hg 2÷. Magos, as well as others (Hayes and Rothstein, 1962; Berlin et al., 1969a) found that tissue distribution was not altered by wide variations in the exposure levels of Hg °. In regard to absorption and distribution, elemental mercury vapor resembles alkyl mercurials rather than inorganic mercury compounds. 7.2. INORGANIC MERCURY

There are a number of publications on the absorption and distribution of inorganic forms of mercury (e.g. HgCl2 and Hg(NO3)2) in animals, but most investigations on man involve mixed exposures to inorganic and organic mercurials; some even involve mercury vapor as well. Localized exposure of the skin of man to high concentrations of mercuric salts may lead to necrotizing reactions, and the oral ingestion of bichloride of mercury results in serious intoxication which may result in death, depending on the dose. Thomson and Russell (1970) have described a case in which systemic and allergic mercury intoxication occurred following silver amalgam dental restoration. Two similar cases of allergic contact dermatitis due to the use of metallic mercury by scientists in their laboratory work were reported by Miedler and Forbes (1968). Miller and Csonka (1968) found that mercury tended to localize preferentially in the kidneys when it was given orally as a solution of mercuric chloride to mice. Swensson and Ulfvarson (1968) showed that in rats given a single injection, mercuric nitrate, like phenyl mercuric hydroxide, had a shorter retention time than did methylmercuric hydroxide. Using radioactive mercuric nitrate (~°3Hg) in rats, Farber and Cember (1969) found that the mercury was preferentially bound to the a2-globulin fraction and to a lesser extent to albumins. This is in contrast to results of in vitro tests in which it was found that a larger fraction of mercury was bound to albumin and the smaller fraction to globulins of plasma. In their studies on the effects of exposure to mercury of workers in several plants, Goldwater and associates (1962) pointed out that there was a reasonable correlation between urinary mercury and blood mercury. In two subsequent studies these workers (Ladd et al., 1963; Jacobs et al., 1963) found consistent correlation between duration of exposure and urinary mercury but not between exposure and blood mercury levels. Actually, according to Kehoe (R. A. Kehoe, 1971, unpublished studies), the correlation between exposure to mercury and urinary levels is quite good if care is taken to reduce variability in sample collection procedures. Ladd et al. (1963) and Jacobs et al. (1963) also pointed out that there was no correlation between urinary mercury levels and the presence of symptoms. According to Battigelli (1960), normal urine contains 0-10 txg/1 of mercury, and Jacobs et al. (1964) suggest that nomal urine values are usually less than 50/~g/l. 7.3. ORGANIC MERCURY

Organic mercurials other than diuretics are of two kinds: phenylmercuric salts and methyl or other alkylmercuric salts. Some comparisons have been made of these forms with inorganic mercurials. Although man has been exposed occupationally and environmentally to organomercurials for a long time, no clear cut information on their absorption and metabolism has emerged from the limited studies done in man. Ladd et al. (1964) reported that phenylmercurials used in several industries have not produced evidence of serious toxicity, which confirmed an earlier report of Massmann (1957). Ladd et al. (1964) could find no consistent pattern of excretion, but they did report a high incidence of contact dermatitis. Alkylmercury compounds are known to be absorbed by man via both the respiratory and gastrointestinal tracts; over 90 per cent of an oral dose is absorbed. Methylmercury may be transformed to other compounds including inorganic mercury and the element in toto is excreted slowly. The biological half life of methylmercury has been estimated to be from 70 to 100 days. The apparent sensitivity of the fetus to alkyl mercurials may reflect an unusual fetal affinity for them, and it is obvious that exposure of women of childbearing age to alkylmercurials should

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be avoided at all cost. Since these compounds have a long biological half life, removal from exposure at the time of pregnancy is an ineffective measure for avoiding fetal damage. Berlin and Ulberg (1963b) found that there was a more pronounced early accumulation of mercury in liver, muscle, and intestines when phenylmercuric nitrate was injected in the mouse than when mercuric chloride was injected. However, in the latter case, mercury accumulated more in the renal cortex, bone marrow, and spleen. When methylmercuric dicyanamide, an agricultural fungicide, was injected, there was pronounced accumulation of mercury in the brain; the concentration in the hippocampus was especially high. Excretion of methylmercuric ion was evident in both bile and urine, and the placenta offered no barrier to this compound. In a related investigation, Takeda et al. (1968a) compared the excretion of mercuric chloride (MC), phenylmereuric chloride (PMC), ethyl mercuric chloride (EMC), S-ethyl mercuric cysteine (EMCys), and m-butyl-mercuric chloride (BMC) when these were injected subcutaneously in the rat. It was found that the alkylmercury compounds (EMC, EMCys, and BMC) were excreted more slowly than were MC and PMC, and each of the former was concentrated in the brain in higher amounts than MC or PMC, PMC being concentrated in the brain in the lowest amount. Takeda et al. (1968b) also found that where ethyl mercuric chloride was given subcutaneously to rats, the metal appeared primarily in red blood cells as S-methyl-mercuric cysteine in the hemoglobin molecule. This form of ethylmercury was stable and was transferred through the stroma with difficulty. Aberg et al. (1969) studied the metabolism of methylmercury nitrate (2°3Hg) in three healthy volunteers. They found that the main excretory route was the feces and that the biological half-life was 70-74 days. The brain retained about 10 per cent of the total radioactive label, the liver had 50 per cent and there was also some measurable long-term retention of methyl mercury, according to the authors' calculations. Norseth and Clarkson (1971) have studied the biotransformation of methyl mercuric ion in rats to understand the preferential excretion of inorganic mercury in feces. They found that when methylmercuric chloride was injected, binding to cysteine in proteins and liberation of Hg 2÷ occurred. The Hg 2÷ was bound to sulfhydryl compounds, including cysteine. When, however, these two forms of Hg 2÷ were excreted in the bile, the S-methyl mercuric cysteine was reabsorbed, while the inorganic mercury was not, thus causing a net concentration of inorganic mercury in the feces. Recently the Rochester group under Clarkson have been investigating the oral administration of resins with free mereapto groups to animals which have received methylmercuric chloride as a method of preventing the reabsorption of the alkyl mercuric moiety. In his review of mercurials as enzyme inhibitors, Webb (1966) has summarized much of our knowledge of the distribution, metabolism, and excretion of mercurials. In addition to the foregoing facts, he has emphasized the following: (a) mercurials are mostly bound to plasma proteins and erythrocytes in blood, and very little is free to enter tissues, which means that effective concentrations for transport are very low at any #oven time; (b) mercurials are excreted in the urine, complexed mainly with thiols such as cysteine; (c) some organic mercurials are split to form Hg 2÷ in the body; (d) Hg* is readily oxidized to Hg 2÷ in vivo; (e) with the exception of brain and kidney levels, there is no relation between tissue concentrations and pharmacologic action; (f) because of the importance of SH groups in many enzyme reactions, very little mercurial is needed to alter vital tissue activities dependent on SH-enzymes. It may be seen from this discussion that there are clear-cut differences in the uptake, distribution, and excretion of mercury metal (particularly the vapor), inorganic mercuric salts, phenylmercuric compounds, and alkyl mercurials. Therefore, in any study careful evaluation of data is needed to assess consistency of response, since different forms of mercury will act differently in vivo.

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8. BIOLOGICAL AND BIOCHEMICAL EFFECTS OF M E R C U R Y COMPOUNDS In his extensive review Webb (1966g) states that 'the early concept of mercurials as nonspecific denaturing and coagulating agents for enzymes has gradually been abandoned in favor of a picture in which definite and often isolatable mercurial complexes are formed under the proper experimental conditions'. Thus at the basis of the toxic action and therapeutic uses of mercurials is the fact that the compounds have specific actions on certain groups, primarily SH groups of enzymes. A discussion of the pharmacologic, toxicologic, or therapeutic activity of mercury compounds therefore requires some specific information and insight into the various normal metabolic processes which may be affected by the mercurials. Inhibition of enzymes by mercurials involves a variety of mechanisms, competitive, non-competitive, uncompetitive, and mixed (Webb, 1966). Enzymes are affected by inorganic and organic mercurials; these include mercuric ion, p-mercuribenzoate ion (pMB), phenylmercuric ion (PM), p-mercuriphenylsulfonate (pMPS), and methylmercuric ion (MM). Mercurial inhibitors resemble other sulfhydryl binding agents such as iodoacetemide in many ways. Enzyme inhibition often depends on the blockage of SH groups, and biochemists use such mercurials as pMB to titrate the SH groups in proteins and other molecules. Examples of enzyme inhibition by mercurials are the following: (a) inhibition of heart lactic dehydrogenase by mercurials parallels the blockage of SH groups (Millar and Schwert, 1963); (b) the inhibition of yeast alcohol dehydrogenase by mercurials develops only after a number of SH groups have been blocked; the most reactive SH groups are not involved in enzyme function (Barron and Levine, 1952); (c) liver cytochrome C reductase inhibition occurs with the blockage of the first SH groups and is complete before all SH groups have been inhibited, which indicates that the most reactive SH groups are associated with the active center of the enzyme (Strittmatter, 1959). These illustrations indicate how mercurials may have specificity of action, and how targeting varies with the different forms of mercury and with their aqueous and lipid solubility. Mercurials can also stimulate enzyme activity, especially at low concentrations; Webb (1966b) gives many examples of this phenomenon. Among the enzymes which are stimulated are Ca2+-activated myosin ATP-ase, which was found by Potis and Meyerhof (1947) to be stimulated by PM, and the dihydrofolate reductase of chicken liver (Kaufman, 1964). Mercurials inhibit enzymes of the electron transport chain as well as oxidative phosphorylation. These inhibitory actions occur at 10-' to 10~M concentrations. The respiratory activity of tissue is inhibited to some extent by mercurials, depending on the substrate used, and the pH of the reaction, as well as on the specific mercurial involved. Sometimes tissue respiration is stimulated; in this regard mercurials do not act like other SH reagents such as iodoacetate or arsenicals. This action may be due to the effect of the mercurial on membrane function or the uncoupling of oxidative phosphorylation. Certain lipid biosynthetic pathways are greatly inhibited by pMB, pMPS, and Hg 2+ at 10 -4 t o 10-5 M concentration. For example, mevalonate transformation to squalene and sterol formation from squalene are sensitive to mercurials (Popjfik et al., 1958, Goodman, 1961). Protein biosynthesis is not particularly sensitive to mercurial inhibition, but porphyrin synthesis is. Thus Lascelles (1966) found that the formation of porphyrin by Rhodopseudomonas spheroides was blocked 100 per cent by 4 x 10-5 M pMB. Purified chelating enzyme is inhibited 75 per cent by 10-'M Hg 2÷ (Labbe and Hubbard, 1961). From these facts it may be suggested that the depressed porphyrin synthesis found with purified systems probably accounts for the depression of hematopoiesis which occurs in chronic mercurialism.

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Another important action of mercury compounds, especially Hg ~÷, is on membrane permeability. The relationship of such action to the pharmacology of mercurials has been reviewed by Passow et al. (1961). They point out that the permeability of yeast cells to dye after treatment with mercury parallels the loss of K ÷ from the cells and that a similar relationship between efltux of K ÷ from yeast cells and the ability to form colonies can be shown. Suzuki et al. (1970) recently showed that the interaction of alkyl mercurials with phospholipid monolayer paralleled the hemolytic action of the derivatives. Thus, the order of each activity for alkyl mercuric chlorides was nbutyl > n-propyl > ethyl > methyl groups. It was also found that these two activities were similar for the phosphates of the ethylmercuric ion, the order of decrease in activity being tri-> di->mono-methylmercuric phosphate. These data and others previously mentioned in this review strongly suggest that the action of mercury compounds on membrane stability, permeability, and function may precede many of the physiological effects of the mercurials. 9. DIURETIC ACTION OF ORGANIC MERCURIALS There is a special class of organic mercurials which have diuretic activity of therapeutic significance. There are many reviews on the nature, use, and mechanism of action of these compounds (Pitts, 1959; Farah and Miller, 1962; Weston, 1957; Evans, 1957), and since a detailed discussion of their pharmacology and therapeutic use will be included in another volume, only a brief mention of these compounds will be made here. T h e main mercurial diuretics have structures which all derive from the following general formula: R--CH2--CH'--CH~----HgX

R can vary widely; it often is in the form R - C - N - and often contains a solubilizing carboxylic acid. Y is usually CH3, CROCH2. X may be either acetoxy, hydroxy, theophylline, chloride, or thioglycollic acid. The last work on the mechanism of action of organic mercurial diuretics has probably not been written; but because of the elegant experiments of Clarkson et al. (1965) much of the heat seems to have gone from the controversy over whether their action is due to the intact molecule or to Hg ~+ which is released in small amounts in the kidney. These investigators showed that in the rat, kidney levels of Hg 2+ correlated with the onset of diuresis. They were able to detect the small amounts of Hg z÷ because of their use of isotopically labeled chlormerodrin and an especially sensitive method for detecting 2°3Hg2+. Kazantzis (1970), in a recent review of the effect of mercury on the kidney, indicated his acceptance of the formation of Hg 2+ in the kidney as the basis for action of all mercurial diuretics. The mechanism of action of the intact diuretic appears to involve concentration within the kidney and release of small amounts of Hg 2+ in the proximal tubule to inhibit resorption of sodium ion. Pitts (1958) has pointed out that only 15-30 per cent of Na + resorption is sensitive to mercurials and that the metabolic disturbance by mercurials must be very small and therefore hard to detect. It appears that this is the situation even at this time. No definite enzymatic or membrane inhibition can be defined as the basis for the diuretic action of mercurials. Some of the problems which still beset the study of the mechanisms of mercurials diuresis are the following: (a) there are great species variations; (b) animal response differs according to water load, ion load, and pH; (c) theophylline-containing diuretics have two points of action the mercurial and the purine; (d) animals and man respond differently to different routes of administration of the diuretic compounds.

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