Effects of arsenic cell metabolism and cell proliferation: cytogenetic and biochemical studies.

Chromosome analysis of lymphocytes from patients who had been exposed to arsenic showed frequent structural and numerical aberrations, even with an interval of decades since the last exposure. The in vitro addition of sodium arsenate induced the same chromosome changes--even to extreme of chromosome pulverizations--upon lymphocyte cultures from healthy subjects. Radioactive incorporation studies showed that arsenate was able to inhibit dose-dependently the incorporation of radioactively labeled nucleotide in RNA and DNA. Beyond that, arsenic blocked the cells in the S- and G2-phase. A general explanation for the inhibitory effect of inorganic arsenic on cell metabolism is the known strong affinity of arsenic to enzymes, especially to those containing sulfhydryl groups.


Introduction
The well-known carcinogenic effect of inorganic arsenic compounds indicates that these substances can directly or indirectly alter the distribution or composition of "genetic" material localized in the chromosome and thereby provide a prerequisite for the origin of atypical cell populations (1, 2). We found massive lesions of the cellular nucleus-even ",chromosome pulverizations''-after in vitro exposure to arsenic in lymphocyte cultures taken from subjects not exposed to arsenic (3). For this reason we studied the problem of whether the lymphocytes from persons who had previously come into contact with arsenic, either in their profession (vine-growers) or for therapeutic reasons (e.g., psoriasis) demonstrated corresponding chromosomal changes.
Supplementary experiments with in vitro administration of arsenate in cultured lymphocytes should

Cytogenetic Studies
Test subjects were 62 patients of the University of Freiburg (i. Br.) Dermatological Clinic. Of these patients, 31 had a history of extensive arsenic contact. In some cases, the last arsenic exposure was more than 30 years previously. The test group consisted of 14 psoriasis patients and 17 vine-growers.
All displayed typical arsenic hyperkeratosis on the palms of their hands and the soles of their feet (4). Several of the patients already had arsenic-induced skin carcinomas excised. The control group (31 subjects) consisted of 14 psoriasis patients and 17 healthy volunteers without traceable history of arsenic contact.
For the chromosome analysis phytohemagglutinin (PHA)-stimulated lymphocyte cultures were prepared according to Moorhead et al. (5). Production of air-dried preparations was followed by staining with orcein diluted in acetic acid (6).

Radioactive Incorporation Test
The preparation of the PHA-stimulated lymphocyte cultures was carried out by use of the techniques described by Moorhead et al. (5). The prep-aration of the culture material for autoradiography and liquid scintillation counting has already been described in detail by Petres et al. (7,8). To the individual lymphocyte cultures from volunteers not exposed to arsenic sodium arsenate was added in concentrations between 0.05 and 100 ,ug/ml culture medium 24 hr before stopping the culture. Cultures from the same subjects prepared in a parallel manner but without the addition of arsenic served as controls.
DNA Cytophotometry Suspended lymphocytes exposed in vitro to sodium arsenate (0.1 to 500 ,ug Na.,HAsO,/ml culture medium) were smeared on a microscopic slide, air-dried, and mounted. One series of preparations was stained with gallocyanin chromatalaun (9). Subsequently, the same preparation was covered with Kodak AR 10 stripping film and exposed for 10 days.
DNA in nonlabeled cells was determined cytophotometrically with an integrating microdensitometer (Barr and Stroud, Glasgow); measurement conditions: objective 100 x, extinction level 0.75, magnification stage 5, wavelength 575 nm; 50 cells from each preparation were measured.

Cytogenetic Studies
The frequency of chromosome aberrations among our subjects in the arsenic-exposed subject group was significantly above that of the controls. This difference is especially evident in chromatid and chromosome aberrations, which exhibit remarkably low spontaneous frequency (Table 1). The number of breaks per mitosis amounted to 0.07 in the arsenic-exposed group and only 0.002 in the controls. ferentiated the arsenic-exposed group from the controls. The aneuploidy is constantly caused by chromosome "deficiency" (Fig. 1). Our results confirm those of our preliminary studies in 1970 (11).

Radioactive Incorporation Test
The experiments show that even relatively low dosages of arsenic cause mild to severe impairment of nuclear division (Figs. 2 and 3). It is remarkable that even "pulverized" chromosomes are able to incorporate in vitro the radioactively labeled thymidine as precursor of DNA (Fig. 4).
Autoradiographic experiments have further shown that the incorporation of :3H-labeled thymidine in the DNA above a dosage of 0.1 g sodium arsenate/ml culture medium is reduced in dose dependency compared with the control preparations. The incorporation of 3H-uridine in the RNA is less clearly inhibited by arsenic (Fig. 5). Scintillation measurements of acid-precipitating material from arsenic-treated cell cultures showed that arsenic inhibits the dose-dependent incorporation of exogenously added 14C-labeled nucleoside in both the DNA as well as the RNA (Fig. 5).

DNA Cytophotometry
DNA measurements of PHA-stimulated lymphocytes demonstrated that under increased arsenic concentrations hyperdiploid cells decreased, i.e., in cells which are in the synthesis and G., phase. At the same time, an increase in hypodiploid cells was ascertained (Fig. 6).

Discussion
The chromosomal aberration rate we observed in vinegrowers and psoriasis patients who had been previously exposed to arsenic was significantly above the value for the control group and above the value reported by Court-Brown et al. (10) for the frequency of occurrence in an average population. It must, therefore, be assumed that the increased number of aberrations is directly related to the history of arsenic contacts (11).
The mechanism which leads to these chromosomal changes is not yet entirely clear. It is, however, presumable that arsenic acts as an impediment to diverse enzyme-systems which interfere with nucleic acid metabolism, especially by interaction with those enzymes which contain sulfhydryl groups (8,(12)(13)(14). The cytophotometrically proven arsenic-produced blocking of lymphocytes in the Sor G.,-phase finds its cytogenetic correlation in the more frequent appearance of endoreduplications, i.e., cells with tetraploid chromosome sets (Fig. 3).
A further site of activity by arsenic on cell metabolism could be that arsenic, as in the mechanism of decoupling of the substrate chain phosphorylation, either incorrectly builds into the nucleotide chains during nucleic acid synthesis or in higher concentrations competitively inhibits the incorporation of phosphorus. The inhibition would then be dependent upon the arsenic phosphorus ratio.
The results of our radioactive incorporation studies are comparable with those of Sibatani (17), who proved that inorganic arsenic compounds Environmental Health Perspectives could inhibit the incorporation of radioactivelabeled phosphorus into the nucleic acid. It is conceivable that the messenger RNA is already altered during its formation and delivers faulty information to the sites of protein synthesis, e.g., enzymes of replication, repair, and formation of precursors (18)(19)(20)(21)(22)(23). Although not statistically significant, the different inhibition-curve progressions of thymidine and uridine incorporation indicate that the DNApolymerase is more severely inhibited than the RNA-polymerase (8). These reactions could provide an explanation for the observed chromosome aberrations both in vivo and in vitro (24,25). If primary cells are damaged as a result of contact to arsenic and subsequently survive, they could then become the stem cells (26) for a pathologic population still detectable many years later in the form of aneuploid cells or cells with marked chromosome anomalies in chronic arsenic intoxication (11).
This impairment mechanism has an effect not only upon lymphocytes but also other cell systems which in chronic arsenic patients manifest themselves in more frequent neoplasias of the skin, liver, and bronchi (27,28). The few leukoses (1) can be explained by a lower sensitivity of the hematopoietic system to the mutagenic agent arsenic.