Toxicological Interactions among Arsenic, Cadmium, Chromium, and Lead in Human Keratinocytes

Abstract

To evaluate health effects of chemical mixtures, such as multiple heavy metals in drinking water, we have been developing efficient and accurate hazard identification strategies. Thus, in this study, we determine the cytotoxicity of arsenic, cadmium, chromium, and lead, and characterize interactions among these metals in human epidermal keratinocytes. Three immortal keratinocyte cell lines (RHEK-1, HaCaT, and NM1) and primary keratinocytes (NHEK) were used. A statistical approach applying an additivity response surface methodology was used to test the validity of the additivity concept for a 4-metal mixture. Responses of the 4 keratinocyte strains to the metal mixture were highly dose-dependent. A growth stimulatory effect (hormesis) was observed in RHEK-1, NM1, and NHEK cells with the metal mixture at low concentrations (low ppb range). This hormesis effect was not significant in HaCaT. As the mixture concentration increased, a trend of additivity changed to synergistic cytotoxicity in all 4 cell strains. However, in NHEK, RHEK-1, and HaCaT, at the highest mixture concentrations tested, the responses to the metal mixtures were antagonistic. In NM1, no significant antagonistic interaction among the metals was observed. To explore a mechanistic basis for these differential sensitivities, levels of glutathione and metallothioneins I and II were determined in the keratinocyte cell strains. Initial data are consistent with the suggestion that synergistic cytotoxicity turned to antagonistic effects because at highest mixture exposure concentrations cellular defense mechanisms were enhanced.

Alloreactive microenvironment after human hematopoietic cell transplantation induces genomic alterations in epithelium through an ROS-mediated mechanism: in vivo and in vitro study and implications to secondary neoplasia

“…Mutant fraction for each experiment was calculated as the number of hyg r colonies corrected for relative to control cell survival as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 21 Treatment conditions were adjusted so that at least 70% of survival was ensured. Induction of GI was expressed by comparing each mutant fraction with the number of hyg r colonies in experiment's internal control.…”

Understanding the human health effects of chemical mixtures.

“…Figure 2 depicts some of the signaling pathways that may be affected by chemical mixtures, and the ways in which they may interact, based on the current literature. Even when we consider a limited number of signaling pathways, the potential not only for additivity and synergism but also for antagonism of toxicity is clear (318). It is apparent that stress-response pathways interact to enhance cellular defense mechanisms and limit metabolism that potentially leads to cellular damage.…”

Characterization of gene expression changes associated with MNNG, arsenic, or metal mixture treatment in human keratinocytes: application of cDNA microarray technology.

“…RHEK-1 cells were also exposed to low doses (9, 11, and 14 ppb) of As 3+ , the metal mixture, or water vehicle controls, continuously for approximately 6 months, or 25 passages; that is, the test chemicals were added to the culture medium at each subculturing. The concentrations of As used corresponded to the LD 2.5 , LD 5 , and LD 10 , as determined in our laboratory for this cell type (11). The lowmixture treatment group was composed of 1, 10, 62, and 33 ppb of As, Cr, Cd, and Pb, respectively.…”

“…Additionally, the activity of a metal in any given tissue is dependent on its speciation and metabolism (6). To further complicate the picture, metals have been shown to interact at multiple levels and, most likely, modify one another's cytotoxicity and/or carcinogenic potential (8)(9)(10)(11). As a result, we are still a long way from a fundamental understanding of the actions of metals or metal mixtures at the cellular level, particularly as they relate to toxic end points.…”

“…Chronic exposure to As leads to skin disorders such as hyperkeratosis and, in many cases, carcinogenesis (12,13). Both As and Cr, a well-known skin sensitizer, have substantial effects on epidermal keratinocytes in vitro and in vivo; these metals have been shown to alter expression of numerous growth regulatory factors, to stimulate cell proliferation at low concentrations, and to inhibit the normal process of differentiation (11,(14)(15)(16)(17)(18)(19). They have not, however, been shown to be directly transforming in this cell type.…”