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4 Metabolism of Arsenic PLANTS Among the many chemical combinations in which arsenic may exist, some readily enter plants and translocate, some in the symplast and some in the apoplast (nonliving cell-wall phase). Plants take up relatively small amounts of arsenic from soils in their natural state and exhibit no symptoms of arsenic injury. Around smelters, high-arsenic soils may be rendered completely sterile and bare of higher plants. Natural waters are usually low in arsenic, and plants reflect this in their arsenic contents. However, some natural waters are very high in arsenic and may thus cause problems for plants (as well as animals). Arsenic in the air in gaseous form has not been known to cause injury to plants. Particles from smelter fumes and smoke may settle out on plants; these may prove toxic to animals or to man, and they may harm plants through the soil. Absorption of Arsenic Compounds Arsenic rarely occurs naturally in the topsoil in quantities toxic to plants. Indeed, all soils contain stance arsenic, and plants have evolved 80

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Metabolism of Arsenic 81 in its presence. In outcroppings of ores high in arsenic, small areas may contain arsenic in toxic quantities. Arsenic uptake by plants is of concern principally around smelters, where arsenic trioxide dust has spread over large areas, rendering the topsoil sterile. With the intro- duction of modern methods of arsenic recovery, cases of soil steriliza- tion by smelter dusts have practically disappeared. Sulfur dioxide, which often accompanies the arsenic trioxide in smelter effluent, has caused more widespread damage. The uptake of inorganic arsenic by plants affects the arsenic cycle. The arsenic concentrates in leaves, which fall to the ground. It is returned to the surface of the soil-at least in some organic, pentavalent form-after the leaves decay. Arsenic is known to be fixed in soils.837 Thus, what arsenic occurs in a mobile form is absorbed out of a deep soil profile, is deposited on the surface, is fixed to varying degrees, and, after oxidation, is subject to leaching by rainwater. Eventually, some of the arsenic originally present in the soil is returned by leaching and takes its original position in the soil and ground water. Some of it also moves in the ground water into springs and streams and is eventually carried into the ocean. Studies of arsenic fixation in soils have indicated that arsenic ab- sorbed by plants must be mainly in the soil solution. Thus, arsenic in water should be available for uptake by microorganisms, algae, and the roots of higher plants. Except for locations around smelters or where the natural arsenic content is unusually high, the arsenic taken up is distributed throughout the plant body in nontoxic amounts, e.g., arse- nic trioxide below 0.02% (dry weight) of foliage or 0.0003% (dry weight) of roots in the case of bindweeds (see Appendix A). These values are very high, compared with those reported by Woolson for the arsenic contents of a wide variety of plants~.O3~.5 ppm. Natural arsenic absorption from the air is negligible. Smelter fumes and dusts may deposit on plant leaves, but there is no evidence that arsenic from this source is taken into plants. Sulfur dioxide in the atmosphere, and more recently constituents of smog, may have de- leterious effects on plants, but not arsenic. Although arsenic may be present in volcanic emission and in some soils, it is rapidly oxidized in air to a particulate form that will not penetrate plant cuticle. Translocation of Arsenic Compounds The application of dilute sodium arsenite solution to bindweed foliage in 1917 resulted in the killing of root tissues and showed that it was possible for toxic compounds to be translocated in plants.306

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82 ARSENIC Two vascular systems in plants are responsible for translocation: the xylem, which transports water and salts absorbed by roots from the soil to the top of a plant; and the phloem, which carries elaborated food materials from green to nongreen parts of the plant. Under conditions of high transpiration, the rate of intake of water by roots, even from an abundant supply, may fall so far behind the rate of evaporation from leaves that a reduced or subatmospheric pressure is built up not only within the xylem conduits, but within all living cells. Under these conditions, if the xylem is cut or broken under a solution, the liquid will be forced into the conduits until the internal pressure equals that of the atmosphere. If the plant is growing in soil in which the moisture is below field capacity, solution will be forced into the cut stem and move throughout the roots, the liquid originally present in them moving osmotically back into the soil. This is a reversal of the normal flow of the transpiration stream. If a solution of a strong acid is applied to the leaves so that they die rapidly, the sap that the cells contain can be forced into the xylem and down into the roots. If arsenic in sufficient quantity is included in such a solution, it too will be injected into the roots, where its toxic action is expressed, and the roots will die. This is the mechanism responsible for translocation of arsenic in the acid-arsenical method. When the tops of plants were cut off at the ground level under sodium arsenite solution, the roots were killed to depths of 7 It (2.1 m) and even more. And if normal plants were sprayed with the acid- arsenical solution and resprayed with water often enough to keep the foliage wet for an hour, depth of penetration of the arsenic was increased. On the basis of the anatomy and physiology of bindweed plants and their distribution in soils, it was explained that the translo- cated acid-arsenical spray could not be used for eradication, because roots at the edges of infestations have available water and hence do not absorb arsenic solution. i79 The authors stressed the influence of tem- perature, incident radiation, humidity, and air velocity on evaporation from leaves. They recommended delaying application until after dark. In years of deficient rainfall, when the soil is moistened to only 3 It (0.9 m) or less, the acid-arsenical method is not effective for killing bindweed, because arsenic does not penetrate below the current sea- son's roots, which are confined to the moist soil layer. The acid- arsenical method might prove useful in eliminating old deep-rooted perennial weeds before the application of a soil sterilization treatment. Sodium arsenite, long used as a general contact herbicide, was not considered to be translocated, probably because, as a contact treat- ment, it was used at a concentration that rapidly destroyed the foliage.

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Metabolism of Arsenic 83 This prevented food movement with which arsenic movement is as- sociated. At lower concentrations, it was combined with acid to hasten penetration; this also rapidly killed the foliage. Rumberg et al.693 compared DSMA at 100 mg/ml with sodium arsenite (4 parts arsenic trioxide to 3 parts sodium hydroxide) at 50 mg/ml; both DSMA and sodium arsenite were labeled with arsenic-76. With soybean plants, they found evidence of translocation of sodium arsenite, but the organic form of arsenic, DSMA, proved to be more mobile. Sodium arsenite injury appears first as loss of turgidity, indicating possible effects on membrane integrity, whereas the organic arsenicals usually cause slowly developing chlorosis with little or no wilting; different enzyme systems may be affected. Rumb erg et al. 693 suggested that the rapid injury from sodium arsenite treatment may be responsible for the lesser transport; the arsenite usually produces injury symptoms within a few hours of treatment, whereas DSMA requires many hours or even days to produce chlorosis. Cacodylic acid is considered to be a general contact toxicant. It is applied in solution to cuts around the base of trees or on foliage. Apparently, its only translocation is apoplastic. DSMA and dodecyl (octyl~ammonium methanearsonate (amine methanearsonate, AMA- equal parts of tetraoctylammonium and tetradodecylammonium methanearsonates) were both shown by Long and Holt to be effective for controlling purple nutsedge Cyperus rotundas) in Bermuda grass (Cynodon dactylon) turf.483 This result can be explained only by translocation from foliage to tubers, a movement that occurs via the phloem. In a later paper, Long, Allen, and Holt showed that those organic arsenicals kill nutsedge tubers if they are applied several times over a 2-year period.482 Long and Holt483 showed amine methanearso- nate to be somewhat superior to DSMA at equivalent rates of application-a result that seems logical in view of the mechanism of herbicide activation described by Crafts and Reiber.~ In further studies on purple nutsedge, Holt et al., 366 using single and repeated applications of amine methanearsonate to shoots of single tubers and shoots of terminal tubers on chains of tubers, found that arsenic was translocated laterally into tubers separated from the treated shoots by up to four tubers. The tuber at the opposite end of the chain from the treated shoots tended to have a higher arsenic content than the tubers between; translocated arsenic tended to be higher in tubers in which active growth was taking place. This is in accord with the source-to-sink nature of food movement; actively growing tubers are active sinks. There was no apparent relationship between the arsenic content of tubers and their ability to produce new shoots, and

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84 ARSENIC there was a tendency for tubers to produce more than one shoot in the regrowth after treatment. Evidently, arsenic treatment altered the apical dominance in tubers that received arsenic. The writers con- cluded that death of tubers after repeated treatments was due to depletion of food reserves, rather than to the concentration of arsenic in the tubers. The variability in arsenic content in killed tubers and the variability in the number of treatments required to kill tubers suggested that failure to sprout is not related to the overall arsenic content of the tuber; some viable tubers contained more arsenic than some dead ones. Interruption of normal oxidative phosphorylation and exhaustion of the food supply resulting from increased sprouting may also have been involved in the ultimate death of tubers. In the repeated-application tests, the arsenic content decreased in tubers from which new rhizomes, tubers, and shoots developed. This indicated retransloca- tion, a property common to phosphate, amitrole (3-amino- 1,2,4- triazole), and dalapon (a sodium salt of 2,2-dichloropropionic acid) in plants. The high arsenic content of terminal tubers and the appearance of chlorosis in untreated shoots confirm this interpretation. Roots and newly developing shoots were not analyzed, and these might account for the loss of arsenic after the initial influx into a sprouting tuber. Translocation of the methanearsonates is indicated in many field studies. For example, Johnsongrass greenhouse studies by Sckerl et al. 7~9 showed rhizome deterioration when 12-in. (30.5-cm) plants were sprayed with DSMA. Kempen et al.4~9 showed that a single 6-lb/acre (6.7-kg/ha) treatment gave better rhizome and regrowth control than 3 lb/ acre (3.4 kg/ha). However, McWhorter found no evidence of rhizome deterioration in his many trials; he ascribed control to top-kill only.535 OnPaspalum conjugatum (sourgrass), Headford suggested that fail- ure of sequential treatments of DSMA to give good results may have been due to relatively inferior transport to the shoot apices.340 On purple nutsedge, Holt et al. 366 showed arsenic accumulation in tubers after foliar sprays. On hardstem bulrush, Hempen indicated that rhizomes showed symptoms. 4~8 Lange's trials on deciduous trees indicated translocation, in that symptoms appeared in new growth and in untreated growth on the trees (personal communication, 1968~. Studies on cotton by Ehman and Baket et al. indicated that arsenic derived from methanearsonates applied to the foliage may ultimately be present in cottonseed.46 229 Rumberg et al. did comparative translocation studies with t76As] DSMA and F76As~sodium arsenite on soybean at 85 F (29.4 C) and found translocation to be greater with DSMA.693 But he was able to recover only 30~0~o of the radioactivity from the DSMA in the foliage,

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Metabolism of Arsenic 85 whereas 85% of the arsenite was recovered. He assumed that the DSMA not recovered may have gone into the roots, which he did not measure. With crabgrass, Rumberg et al.693 found translocation and toxicity with DSMA greater at 85 F (29.4 C) than at 60 F (15.6 C) on crab- grass and soybean; on the latter plant, movement was more basipetal (to the basal stem) than acropetal (to the opposite primary leaf and trifoliate leaf). The toxicity of sodium arsenite and cacodylic acid was not affected by the temperature difference. Sckerl and Frans indicated that t~4C]methanearsonic acid (~14C]MAA) was absorbed by Johnsongrass roots in significant amounts in 1 h and moved throughout the plant in 8 h.7~7 Spot applications of the acid formulation to foliage resulted in acropetal movement within 8 h and movement throughout the plant within 24 h. Duble et al. studied the translocation of two organic arsenicals in purple nutsedge.2~6 DSMA and AMA were the forms used, and tracers containing carbon-14 were applied to greenhouse plants. Chromato- graphic tests on extracts from t~4C]DsMA-treated plants indicated that the compound was not readily degraded; the i4C-As bond ap- peared to remain intact, although some t~4C]carbon dioxide was found several days after treatment. Comparison of the retardation factors (Rf) of plant extract-DsMA with that of standard [~4C]DsMA suggested that a plant extract-DsMA conjugate might have been formed; the values for extract and standard solution were 0.59 and 0.66, re- spectively; only one spot was found in each case. Over 85% of the material applied to the plant remained in the treated shoots. DSMA moved both acropetally and basipetally in single leaves, and such movement was not influenced by relative age of the leaf. The writers reported that both DSMA and AMA are moved symplastically and apoplastically a property that they share with amitrole. Carbon- 14 distribution in an untreated shoot appeared to be very similar to that reported by Andersen~5 for amitrole distribution in the same plant. It seems apparent from those results that translocation in nutsedge follows a source-to-sink pattern and that the amount of arsenic moved into a tuber depends very much on the sink activity of that tuber. Duble et al. found that actively growing terminal tubers in a chain accumu- lated arsenic, whereas intermediate and dormant tubers did not.2~6 Thus, the arsenic content of a tuber may not serve as an index of the lethality of a treatment; the effects of the initial impact of arsenic on later growth activity may be the critical factor in lethality and continu- ing transport of arsenic to growing roots. Other tubers and shoots may mask the effects of the arsenic content, as determined by analysis at any time.

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86 ARSENIC McWhorter found no evidence of translocation of DSMA in Johnson- grass in field and greenhouse tests.535 Sckerl and Frans, by contrast, found that FI4C]MAA was both xylem- and phloem-mobile in Johnson- grass and cotton.7~8 Root uptake of this labeled arsenical by Johnson- grass from nutrient solution was rapid, and translocation into all portions of the plant took place within 4 h. Apoplastic movement in both plants was more rapid than symplastic movement; symplastic movement was more rapid in Johnsongrass than in cotton. 7i 6 As Duble et al. found, chromatography of extracts from Johnsongrass revealed values that differed from those of treated plants and standard EI4C]MAA solutions; Sckerl and Frans suggested complexing with sugars, organic acids, or both. When amino acid fractions were prepared from methanol extracts of both plants, an MAA metabolite with a positive ninhydrin reaction was found in the Johnsongrass fraction. Comparing Rf values, the authors suggested a complex with histidine or one of its analogues. Amino acid accumulation was noted in Johnsongrass as a result of MAA treatment, and the authors suggested that the MAA metabolite may block protein synthesis or some other biosynthetic pathway. Wilkinson and Hardcastle produced radioactive arsenic (arsenic -76) by neutron activation and determined concentrations of this radioisotope in new (unsprayed) and old (MsMA-sprayed) cotton leaves and in the soil from beneath the plants; MSMA treatment rate was 2.24 kg/ha.86, By using long counting periods, they were able to extend the lower limit of detection to 5 ng of arsenic. Table 4-1 presents the results TABLE 4-1 Arsenic Content of Leaf and Soil Samples Taken from Field-Grown Cotton Treated with MSMA at 2.24 kg/haa Arsenic Content, ,ug/g New Leaves Old Leaves No. Treatments (Unsprayed) (Sprayed) Soil 0 0.05 0.17 3.75 1 0.13 0.45 8.25 2 0.95 1.05 8.35 3 0.23 o.go 7.50 4 3.15 17.30 10. 15 5 4.80 41.10 4.00 6 10.30 30.25 1 1.20 "Derived from Wilkinson and Hardcastle.H6'

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Metabolism of Arsenic 87 of the analyses. Differences in arsenic content of young untreated leaves from the two increments of MSMA application (i.e., one to three and four to six applications) may be due to dilution of arsenic content by continued growth. The increasing arsenic content in new unsprayed leaves also indicates the presence of translocatable arsenic from MSMA- treated leaves. Kempen has made a study of MSMA in plants, principally Johnson- grass, using both detached leaves and whole plants.4~7 He found that relatively high temperature, 35 C? and light, 2,800 ft-c (30,128 lx), produced SOYo necrosis of rhizomatous Johnsongrass foliage in less than a day, whereas lower temperature, 15 C, and light, 320 ft-c (3,443.2 lx), required 12 days to give the same results. Regrowth from rhizomes showed similar trends, indicating that the arsenic had trans- located into the rhizomes. Translocation of the label from leaf-applied ti4C]MsMA was primarily acropetal in the xylem, but small amounts also moved basipetally, proving that this organic arsenical is phloem-mobile. Within a week, the arsenic was transported from a treated mature leaf into the leaf base and sheath, to meristematic regions, and to roots and rhizomes; this indicates symplastic move- ment. Presence of the methanearsonate was detected by autoradiog- raphy and by counting. Sachs and Michael found cacodylic acid and MSMA to be transported about equally from the leaves to the terminal buds and expanding leaves of bean plants.695 There was no indication that either was demethylated or reduced to a trivalent compound. With ti4C]MsMA, Sachs and Michael found that about 40~o of the carbon-14 and arsenic recovered was bound to another molecule to form a ninhydrin-positive complex.695 Sckerl and Frans7~8 reported this ninhydrin-positive com- plex, as well as a sugar complex, and postulated that such a complex may block a specific biosynthetic pathway and that this blockage may account for the herbicidal activity. Duble et al. noted that DSMA was almost completely complexed with some plant component and that the complex was the mobile form of the herbicide.2~7 They also found that less than O.l~o of the carbon-14 applied as DSMA was metabolized and given off as volatile carbon-14 10 days after treatment. They con- cluded that the carbon-arsenic bond was stable in plants. However, von Endt et al. 827 found this carbon-arsenic bond to be rapidly broken in soils, which suggested that the bond is subject to attack by some biologic systems. In studies with ~ 4C-labeled MSMA, MAA, and DSMA, Keeley and Thullen found little translocation of these herbicides from cotyledons to developing leaves of co.tton seedlings.4~4 An exception was noted

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88 ARSENIC where [14C]MsMA was applied at 13 C; there was little contact injury of the treated cotyledons, and the terminal leaves were well labeled. With the same three herbicides in studies on purple and yellow nutsedge, Keeley and Thullen found the yellow species to be more susceptible; the yellow species absorbed and translocated more of the ~4C-labeled tracers than did the purple.4~34~5 In chromatography of plant extracts and standards, there was less than 5% variation; nut- sedge plants did not readily metabolize these arsenicals in 72 h. Surfactants have been found to increase the penetration and translo- cation of the organic arsenical herbicides. The principal manufacturer of the methanearsonates formulates mixtures containing tested surfac- tants. Apparently, anionic and nonionic surfactants are satisfactory with these materials. A summary of the above discussion is presented in Table 4-2. In almost all cases, the arsenicals are translocated either upward or downward. The rate and direction of movement vary according to plant species and chemical. The degree of response may be temperature- dependent. A metabolite may be formed in susceptible species, but the formation is apparently not necessary for efficacy. MICROORGANISMS The sources of arsenic available to animals and man are naturals (such as water, plants, and animal tissue) and synthetical (such as agricultural chemicals, industrial wastes, and drugs). The natural sources of arsenic are modified by bacteria, molds, and algae, and they will be discussed from this point of view. Bacteria It has recently been shown527 that Methanobacterium (MoH strain) can reduce and methylate arsenate to dimethylarsine (cacodyl hydride). The medium must contain methylcobalamin as a methyl donor and adenosine triphosphate in a hydrogen atmosphere. The formation of the alkylarsine was detected by its odor and by aerating into 2 N nitric acid to oxidize it to cacodylic acid. With t~4CH3]cobalamin and t74As~sodium arsenite, a double-tagged compound was isolated whose ratio of ti4C]methyl to arsenic-74 was 2.0: 1. When cacodylic acid was used as a substrate, the alkylarsine was formed without the need of a methyl donor. The presence of an excess of sodium arsenite inhibited the production of the alkylarsine, the reaction producing

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Metabolism of Arsenic 89 methanearsonic acid. The use of formal oxidation numbers for ar- senic in Figure 3 of Wood's article796 can be misleading, in that there is no actual change in the valence of arsenic, as explained by Zingaro.892 It should be noted that dimethylarsine was not positively identified, because both cacodyl (tetramethyldiarsine) and cacodyl oxide, which have a strong garlic odor, could have been formed and would have the same methyl: arsenic ratio. The recent sugges- tion796 that such a reaction in stream sediments could be hazardous to fish is without foundation; methylarsine decomposes to methane- arsonic acid in the presence of oxygen, 658 and the latter compound is less toxic than sodium arsenite. The necessity for an anaerobic atmosphere greatly limits the possibil- ity of this reaction in soil. Such conditions may be present in the rumen of the cow, and there is evidence that both arsenate and arsenite can be methylated by rumen flora. The reverse procedure, the demethylation of MSMA in soil, has been reported.827 When t~4C]MsMA was added to loam, 1.7-10% of the compound was degraded, yielding t~4C]carbon dioxide and arsenates. This did not occur in soil sterilized by heat. A fungus and two actino- mycetes isolated from the soil degraded 3, 13, and To of MSMA added to a substrate. The bacterial species similarly isolated degraded 20~o of the MSMA to arsenate; this changes the arsenical to a more toxic form. Fungi and Molds As early as 1815, cases of arsenic poisoning were reported to have been caused by wallpaper containing such arsenic compounds as Scheele's green (cupric arsenite) and Schweinfurt green (copper acetoarsenite, Paris green). The mechanism was first thought to be the ingestion of particulate material from the paper; but, when poisoning occurred with fresh paper, that theory wars abandoned. Gmelin (cited in Challenger et al. }39) was the first to report that rooms where symptoms occurred had a garlic odor, and he ascribed it to a volatile arsenic compound produced by molds on damp arsenic-pigmented wallpaper. Numerous investigations attempting to identify the chemical nature of this volatile arsenical have been reviewed by Cha'lenger,~35 i36 whose own research established the chemical structure of the compound. Gosio used pure cultures of bacteria and fungi on a potato medium and found that, although no bacteria produced a garlic odor, a mold, Penicillium brevicaule (formerly Scopulariopsis brevicaulis), was very active. He analyzed the gas formed and concluded that it was an alkyl

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108 ARSENIC Distribution of Arsenic in the Body The most complete study of the distribution of arsenic in animals and man resulted from studies with radioactive isotopes of arsenic. In 1942 Hunter et al. 376 injected [74AsUpotassium arsenite subcutane- ously into rats, guinea pigs, rabbits, chimpanzees, a baboon, and leukemic humans and analyzed their tissues and body fluids. With the exception of the rat, in which the arsenic concentrates in the red l food cells, the arsenic is generally distributed to all tissues, with the largest total amount going to the muscles. Most of the arsenic was excreted by the kidneys; excretion was essentially complete in 6 days, with only a trace appearing in the feces. Arsenic does not pass into the spinal fluid of humans, but small amounts were found in apes. Lowry et al. 488 fractionated various tissues from this experiment and studied the acid-soluble lipid and protein fractions for arsenic and phosphorus content. They also separated liver protein into different fractions, including nucleoprotein. Most of the arsenic was in the protein fraction, a small amount in the acid-soluble portion, and a trace in the lipid fraction. The nucleoproteins did not take up more arsenic than other proteins, and there was no evidence that phosphorus was displaced by arsenic in tissues. Duc off et al. 220 used t76As~sodium arsenite on rats, rabbits, mice, and man, studying their excretion rates and tissue distribution patterns. The rat retained most of the arsenic in the red blood cells, with smaller concentrations in the spleen, heart, lungs, kidneys, and liver. In rabbits, the arsenic was lowest in the blood and highest in liver, kidneys, and lungs. The most interesting results are shown in Figures 4-3 and 4-4, which compare the relative excretion rates in rat, man, and rabbit and the blood concentrations of arsenic in rat, man, rabbit, and chicken. Lanz et al.452 studied the absorption, distribution, and excretion of arsenic-74 injected intramuscularly in the rat, dog, cat, chick, guinea pig, and rabbit. The accumulation of arsenic in the tissues after 48 h showed that less than 0.27% was stored in the organs studied in all species except the rat and cat, which stored 79~O and 5.6%, respectively, in the blood. They studied the distribution of arsenic in rat blood and found that none was bound in the plasma proteins, a trace in cellular ``ghosts," and most of it in hemoglobin from which it could not be removed by dialysis. Arsenic was rapidly excreted in the urine as inorganic compounds, with lO-15% of the arsenate being reduced to the trivalent form. Their analytic method required the precipitation of

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Metabolism of Arsenic 111 O 100 c, L1J ~ 80 A LO o Z 60 c: 111 u' z 40 o - LL x 20 UJ - Rabbit - ! l l - - _ t / ! _- _ Patient GR _ ._ _ ~ - -I ~~ ~ Katie, - Rats 0 24 48 72 ]6 HOU RS AFTE R I NJECTION FIGURE 4-3 Excretion of arsenic-76. Cumulative excretion from the time of injection is expressed as percentage of the adminis- tered dose. Reprinted with permission from Ducoff et al. 220 109 the arsenate as magnesium ammonium arsenate; the filtrate was as- sumed to be trivalent arsenic (which is highly questionable). Mealey et al. 536 studied the distribution and turnover of radioactive arsenic after intravenous injection into man. The isotope used, arsenic-74, was 90% in the trivalent form and was given as the sodium salt in a dosage of 2.3 mCi/70 kg. The different rates of urinary excretion indicated that the arsenic was distributed into three com- partments, as shown in Figure 4-5. Clearance rates for each were calculated as the percentage of the total dose per hour, giving 25%/h for compartment I, 2.5%/h for II, and 0.3%/h for III. The proportion of pentavalent arsenic in the urine showed a steady increase until the fourth day, when it remained constant at 75%.

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110 ARSENIC 100 L So 20 1 0 a 2 At LO 1 1 _ ~ _~ _ RAT win 4c ~-\ MAN \ \ N"~" ~ " RABBIT '\ " _ HICKEN 24, 48 72 96 TIME IN HOURS FIGURE 44 Arsenic-76 in whole blood. Concen- tration of arsenic-76 per gram of blood at a particular time is expressed as percentage of the administered dose per gram of body weight. Reprinted with per- mission from Ducoff et al. 220 The low rate of excretion of arsenic in the rat is probably due to fixation of 80-90~o of it in the hemoglobin of the red cell, which must break down before the arsenic can be released. The high content in blood also makes it difficult to get true tissue values in such organs as the spleen and liver. In an attempt to find another small-animal model for man, Peoples625 studied the distribution of arsenic in rats, guinea pigs, rabbits, and hamsters that were fed arsenic trioxide; the results are shown in Table 4-15. The hamster appears the animal of choice, with the rabbit also a good possibility. The relationship between arsenic ingestion and urinary arsenic has led to its use in measuring industrial exposure. The importance of /

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Metabolism of Arsenic 111 checking the diet for the intake of seafood has been reported.708 It is interesting that a wide variety of fish, crustaceans, and mollusks caused increases of up to 10 times the normal content lasting for over 20 h. No symptoms were reported at concentrations that would have caused serious poisoning if the substance ingested had been sodium arsenate. The chemical nature of the arsenic compound is unknown, but it is probably organic. With the helium-arc method, 99 the urine of four subjects was analyzed for As(III), As(V), methanearsonic acid, and cacodylic acid; the results are shown in Table 4-16. The average excretion of 66~o of the arsenic as DMA is striking, especially because the subjects were from Florida and likely to eat seafood. The relationship of industrial exposure to seafood intake should be analyzed with this method. The distribution of arsenic in the cow has been studied with a variety of compounds in nontoxic doses.625 The feeding of arsenic acid to cows at 0.05-1.25 mg/l~g of body weight for 8 weeks did not increase the arsenic content of the milk, but it did increase the arsenic in the tissues. Urine was the main pathway of excretion, which was very rapid-the urine was free of arsenic 2 days after arsenic administration was 100 10 In o ~5 TO - UJ ~ 1.0 at o LL a: c' o.1 x .- L.L. -J.G. v -R.P. O -E.H. O - D.D. v o _ art v 0 0 0 0 0 0 0 - _ 0 01 ) 20 40 60 80 100 120 140 160 180 200 220 240 260 280 TIME AFTER INJECTION (hours) 0 0 0 0 1 1 1 1 ~ I 1 1 1 FIGURE 4-5 Rate of urinary excretion of radioarsenic in five subjects. Reprinted with permission from Mealey et al. 536

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Metabolism of Arsenic TABLE 4-16 Analysis of Human Urine Samples for Arsenic Compoundsa 113 Methane arsonic Caca As(lII) As(V) Acid Acid Total, Subjectppb 56 ppb To ppb 5! ppb To ppb Ma age 28<0.1 0.84 8.1 0.61 5.9 8.9 86.5 10.4 M age 275.1 20 7.9 30 2.5 9.7 10.4 40.2 25.9 M, age 42<0.5 - 2.4 7.8 2.4 8.1 25.2 84.0 30.0 F. age402.4 10 4.3 18 1.8 7.6 15.5 64.5 24.0 Average1.9 - 3.9 - 1.8 - 15.0 22.6 aDenved from Braman.~9 The results are given in parts per billion (ppb) as arsenic. The precision of individual runs is + logo relative. or +0.1 ppb for small sample sizes. Four other samples not completely analyzed gave: methanearsonic acid. 3.6 + 2.4 (S.D.) ppb; and cacodylic acid, 15.5 + 6.8 (S.D.) ppb. discontinued. The valence of urinary arsenic was determined; pentava- lent arsenic was the only form found. A similar study with sodium arsenite and cacodylic acid yielded essentially the same results (Peoples, unpublished data). When MSMA was fed to lactating cows, the blood concentration of arsenic rose, but the milk concentration remained low.627 These results support the conclusion that there is a blood-mammary barrier to arsenic. The most likely explanation is that an active trans- port mechanism is saturated at normal plasma concentrations. Alipha- tic organic and inorganic arsenic must use the same transport mecha- nism. Toxic doses of arsenic do not break down the barrier to arsenic. Marshall gave Jersey heifers lead arsenate in their feed at 12.95 mg/lOO lb (0.285 mg/kg) of body weight for 126 days and noted no values in the milk above the control value of 0.05 ppm. The same results were obtained by Fitch,254 who fed a heifer 0.3428 g of arsenic trioxide daily for 3 days without increasing the arsenic content of the milk. In these acute studies, the skin and hair were found to have low concentrations, indicating slow uptake. Dubois et al.2~9 showed that exposure to arsenic in atmospheric dust can give fallacious results up to 243 ppm. Washing in detergent reduced all values to 3.0 + l .O ppm. Lander et al. 449 examined hair in arsenic poisoning cases and found a wide range of concentrations, 3.0-26.0 ppm. These values were reached a short time after exposure, and the concentration in hair near the scalp was often no higher than that in hair near the tip. The

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114 ARSENIC difference was thought to be due to contamination from sweat. The same situation was found in the nails. Perkons and Jervis632 used neutron-activation analysis on human hair and reported the frequency distribution of 12 elements, including arsenic, as shown in Figure 4-6. The range of arsenic concentration of 1-5.5 ppm is in accord with the concentrations in washed hair found by Dubois et al. 2~9 Apparently, the value of hair as a reliable indicator of chronic arsenic poisoning is open to serious question (see Chapter 61. The valence of arsenic in water, food, and body tissues is largely unknown, not because of its lack of importance, but because of the technical difficulties involved. The method of Crawford and Storey,~84 in which ethyl xanthate is used to extract trivalent arsenic, has worked well in the hands of Ginsburg,29i who used it in renal clearance studies of arsenite and arsenate in dogs. Ginsburg found that arsenate is reabsorbed in the proximal renal tubule and is reduced in part to arsenite, which then appears in both urine and blood. This is the only evidence of a reduction in valence in a living animal. Winkler868 analyzed the livers of rats that had been fed sodium arsenite and found that most of the arsenic was pentavalent. Livers of rats fed sodium arsenate and sodium arsanilate contained only pentavalent arsenic. With Winkler's method, Peoples625 found only pentavalent arsenic in the urine of cows fed arsenic acid. The arsenic content of neoplasms has received scant attention, although arsenic is often listed as a carcinogen, particularly in books on dermatology (see Chapter 6~. Domonkos209 reviewed studies on the arsenic content of normal skin, skin that was pigmented by exposure to arsenic, and keratoses. Most of the reports indicated normal or low concentrations of arsenic in the lesions, compared with nearby normal skin. He used neutron activation to determine the arsenic in skin and epitheliomas in humans with and without a history of arsenic ingestion. The arsenic content of skin samples varied from 0.15 to 95 ppm when taken from the same patient, and that of four epitheliomas in one patient, from 0.22 to 4.1 ppm. The values for skin vary from 0.1 to 5.0 ppm, regardless of a history of arsenic ingestion. He concluded that arsenic determination in skin lesions was of no value. There are some data on other types of tumors. Ducoff et al.220 administered sodium arsenite to a person with a parotid tumor; the tumor took up less arsenic than the liver and kidney and the same amount as most of the other organs. Ducoff et al. also studied the uptake in mice inoculated with Jackson-Brues embryoma and lym- phoma. The concentrations in the tumors and organs were too variable

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116 ARSENIC to show a significant trend. The liver: spleen: kidney arsenic ratios were different in normal and tumorous mice. Hunter et al.385 injected L74As~potassium arsenite into two leukemic humans and found that the tissue distribution resembled that in the guinea pig. There is no evidence from these data that neoplasms have any peculiar ability to store arsenic. Dermal Absorption of Cacodylic Acid in Man In 1972, the forestry workers in north central Washington applied MSMA and cacodylic acid into the cambium layers of trees.78~ The purpose was to thin the trees and thereby ensure a better stand. The technique resulted in wetting of the workers' clothing, and many developed a garlic breath odor. Urinary arsenic studies were made on a six-man crew over a period of 9 weeks, samples being taken on Monday and Friday of each week. Each man used a different proce- dure, as follows: (1) a control, hack squirt tool with (2) MSMA or (3) cacodylic acid, injection hatchet with (4) MSMA or (5) cacodylic acid, and (6) MSMA with injector tool. The difference between the two chemical means and the method means was not significant at the 5% level. The control was significantly lower than all chemical treatments, with Monday values being significantly lower than Friday values. The values plateaued or dropped during the study, indicating that the chemicals did not accumulate. There were no health problems related to the compounds used. Further studies were made by Wagner and Weswig on forestry workers who were exposed to cacodylic acid over a 2-month period.832 An attempt was made to correlate blood and urinary arsenic concentra- tions with degree of exposure without much success. The urinary values were markedly increased, but did not reach the heights of the previously mentioned study. They attributed the odor to arsine; it is highly unlikely that arsine caused the odor, because it is so toxic. When cacodylic acid was commonly used as a tonic, the odor on the breath was reported to be caused by tetramethyldiarsine (cacodyl).750 Suff~- cient amounts of arsine to give the breath an odor would be lethal. As would be expected, none of the workers complained of ill health, be- cause the amounts of cacodylic acid absorbed were less than the dose formerly prescribed as a drug, or 30 mg/day.