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Humic acid (HA) has been proposed as factor in the cause of Blackfoot disease (BFD) among individuals who live along the southwest coast of Taiwan. In this study, the interaction of the synthetic humic acid, made from catechol, with sodium arsenate (As(V)) was investigated and assessed with respect to damage to human red blood cells. HA is characterized as phenolic and phenolic carboxylic polymer structures containing both -COOH and -OH as their main functional groups. HA and As(V) alone are able to hemolyze 60-100 and 5-20% human red blood cells at concentrations of 50-300 microg/ml and 5-100 mM, respectively, after 6 h. HA is shown to be relatively ineffective in causing ATP depletion of red blood cells. For organometallic complexes composed of HA-As(V) the inhibition effect of EDTA was completely abolished and the use of the triple complex HA-As(V)-EDTA resulted in an enhancement of hemolysis. HA caused lipid peroxidation in a concentration- and time-dependent manner. However, HA-As(V) and As(V) decreased lipid peroxidation. These results indicated that HA initiates oxidative stress on red blood cells and this results in their dysfunction. HA-chelated high-concentration metal complexes inhibited the structures containing the main functional groups involved in decreasing hemolysis, and, thus, HA may be a significant factor in the etiology of BFD.

Despite potential differences in the mechanism and potency of toxicity between the two common oxidation states of arsenic (As(III) and As(V)), assessments of the risk from inhaled arsenic generally ignore the oxidation state of inorganic arsenicals. Differences between potency and toxicity of As(III) and As(V) were evaluated by determining alteration in function of pulmonary alveolar macrophages (PAM) following in vivo and in vitro exposure to soluble arsenic. Male Sprague-Dawley rats were used throughout. One day following intratracheal instillation of 1 mg/ml (as arsenic) of either sodium arsenite (As(III)) or sodium arsenate (As(V)), PAM were lavaged and analyzed for alterations in superoxide (O2−), prostaglandin E2 (PGE2), and tumor necrosis factor (TNF-α) production. There were no differences in bronchoalveolar lavage fluid PGE2 or TNF-α. PAM lavaged from As(V)exposed animals showed significant increases in O2− production. In vivo exposure to either oxidative form of arsenic decreased basal and lipopolysaccharide (LPS)-induced release of TNF-α production by PAM, but did not suppress LPS-induced production of PGE2. To test the direct effects of arsenic on PAM function, PAM were lavaged from control animals and exposed, in vitro, to either arsenical for up to 24 hr to concentrations of 0.1 to 300 μg/ml arsenic. Doses used were not cytotoxic to PAM, since LDH release was not significantly increased, even at the highest dose. Significant dose-dependent inhibition of O2− production was only evident after 24 hr exposure to arsenicals. As(III) was more potent than As(V), inhibiting O2− at concentrations as low as 0.1 μg/ml compared to 1.0 μg/ml of As(V). Suppression of LPS-induced release of TNF-α also occurred at lower concentrations of As(III), 50% inhibition at 0.15 μg/ml, compared to As(V), 50% inhibition at 1.8 μg/ml. While As(II) exposure had no affect on PGE2 production, As(V) caused inhibition of LPS-induced PGE2 production at concentrations above 1.0 μg/ml. Differences between As(III) and As(V) indicate that different mechanisms and/or potencies exist between the two arsenic species. Arsenic-induced alteration in PAM function may compromise host defense against infections and alter immune surveillance.

The response of human and bovine peripheral blood lymphocytes to PHA stimulation was measured in the presence of low concentrations of sodium arsenite and sodium arsenate. In bovine lymphocytes, 41% augmentation of the response occurred at 10(-6) M arsenite with a return to the normal response at 2.5 X 10(-6) M. Complete inhibition of mitogenesis occurred at 6 X 10(-6) M. In the presence of sodium arsenate, similar results were obtained but at the higher concentrations of 2 X 10(-5) (for 57% augmentation), 5.2 X 10(-5), and 1.9 X 10(-4) M, respectively. The possible significance of these findings in view of the known relationship between chronic arsenicalism and human skin cancer is discussed. It is suggested that arsenic compounds may, by potentiating mitogenesis, increase the possibility of errors in DNA replication, some of which could be potentially carcinogenic. Additionally, interference with the immune response could enable potentially cancerous cells to escape immune surveillance.

Whole embryo culture techniques were used to test the effects of sodium arsenate on the rates of DNA, RNA, and protein accumulation. Rat embryos were isolated on the 10th gestational day (sperm day = Day 0) either 4 or 24 hr after maternal injection. The cultivation period was for 24 or 42 hr. DNA, RNA, and protein values were not significantly different for in utero treatment 4 hr prior to dissection. For embryos treated in utero 24 hr prior to dissection, DNA, RNA, and protein levels were significantly lower at the beginning of cultivation and after 24 hr in culture. By 42 hr, DNA and RNA values were still significantly reduced. A number of morphologic differences were also noted following cultivation for embryos treated in utero 24 hr prior to dissection. These include a significant number of embryos failing to (1) rotate to a ventroflexed position, (2) have a closed anterior neuropore, (3) establish a visceral yolk sac circulation, and (4) fuse the allantoic sac in placental formation. It is concluded that sodium arsenate takes longer than 4 hr to affect the embryo and that a 24-hr in utero exposure is adequate to initiate teratogenic responses. The abnormal persistence of a definitive allantoic sac after culture may be indicative of abnormalities in urogenital system formation. The effects of sodium arsenate seem to be related to proliferative embryonic cell requirements with some insult recovery of the embryo possible.

We have determined the concentration, availability, and chemical status of radiolabeled arsenic in the blood of pregnant hamsters at the beginning (morning of Day 8) and the end (morning of Day 9) of the critical period of embryogenesis. Hamster dams were exposed to teratogenic doses of arsenate by means of osmotic minipumps implanted on the morning of Day 6 of the gestation period. Whole blood arsenic concentrations were the same for 48 and 72 hr postimplant. The arsenic concentration of plasma equaled that of red cells. Plasma arsenic was not bound to macromolecules and had the same chemical status 48 and 72 hr postimplant. Arsenate was the dominant form (67% of the total). However, the presence of dimethylarsinic acid and arsenite indicates that the pentavalent species was metabolized. Red cell arsenic was bound to macromolecules in the cell sap. Seventy percent of red cell sap arsenic was dialyzable 48 hr postimplant, but only 56% 72 hr postimplant. Arsenate was the dominant dialyzable red cell species on Day 8 and arsenite was the major dialyzable form on Day 9. Our findings demonstrate a relationship between the maternal blood concentration and chemical status of arsenic and the presence of malformations resulting from a constant rate exposure of pregnant hamsters to arsenate via the osmotic minipump.

The absorption, biotransformation, and tissue retention of arsenic following intratracheal and oral administration of 74As-labeled sodium arsenite, sodium arsenate, arsenic trisulfide (suspension), and lead arsenate (suspension) have been studied in hamsters, and correlated to the in vitro and in vivo solubility of the compounds. After intratracheal instillation, the clearance of 74As from the lungs was positively correlated to the in vivo solubility. Less than 0.1% of the sodium arsenite and sodium arsenate was retained in the lungs after 3 days, compared to 1.3% of the arsenic trisulfide particles and 45% of the lead arsenate particles. The latter showed a very low solubility both in vivo and in vitro. In general, orally administered arsenic had a shorter biological half-life than intratracheally administered, especially when given in the form of arsenic trisulfide or lead arsenate particles, which seemed to be absorbed to only 20-30% in the gastrointestinal tract. Reduction, oxidation, and methylation of arsenic varied to a great extent with the arsenic compound and the route of exposure. Trivalent arsenic was methylated to a greater extent than pentavalent and less soluble compounds (suspended particles) more than dissolved compounds. The trivalent arsenic compounds caused higher concentrations than the pentavalent in the upper gastrointestinal tract but not in other tissues.

Similar malformations of the central nervous system including encephaloceles and exencephaly are induced by treatment of pregnant hamsters with sodium arsenate or hyperthermia on the eight day of gestation. These two treatments are synergistic in that combinations of minimal teratogenic levels of each cause a marked increase in fetal resorptions and the frequency and severity of developmental malformations as compared to separate treatments. The possible relationship of maternal hyperpyrexia and environmental teratogens to congenital malformations is discussed.