Cadmium is a well-known toxicant that is continuously introduced into the environment. Environmental exposure to cadmium is a substantial public health concern. Recent studies suggest that incidences of cadmium-associated disease are escalating in populations exposed to low levels of cadmium 
. To more fully understand the relationship between cadmium and disease, it is imperative to understand the mechanism of cadmium-responsive transcription, under both adaptive and toxicological conditions. Toxicological effects at low-levels of exposure are prevented or repaired by altered expression of multiple stress-response proteins and their cognate genes. Alterations in gene expression have been observed in multiple systems at cadmium concentrations below those leading to measurable toxicological responses. In vitro
exposure of HeLa or CCRF-CEM cells to non-toxic concentrations of cadmium affects the expression of ~60 and 20 genes, respectively 
. Treatment of mice with non-toxic doses of cadmium causes the differential expression of 22 genes 
. Likewise in C. elegans
, ~100 genes are differentially expressed following cadmium exposure under conditions that do not induce a general stress response 
At toxic concentrations of cadmium, the transcription of hundreds of genes is affected. Among the hundreds of cadmium-responsive genes, many of the cognate regulatory pathways have been identified. These pathways include MAPK, p53, NRF2, Protein Kinase C, casein kinase 2, and CaMK II 
. Although regulatory pathways have been identified, the molecular mechanisms by which cadmium initially activates these pathways to elicit specific transcriptional changes have not been defined.
One hypothesis that addresses how cadmium activates intracellular signaling pathways proposes that the metal modulates the level of [Ca2+
. Thus, calcium could be viewed as a second messenger that mediates cadmium-responsive transcription. While several mechanisms have been proposed by which cadmium may alter [Ca2+
, the effects of cadmium on calcium signaling remain ambiguous. This ambiguity may be a consequence of technical approaches traditionally used to investigate calcium-mediated signaling processes. Specifically, there are potential problems in the interpretation of data from studies in which BAPTA or BAPTA-based fluorescent calcium indicators are used when examining the consequences of cadmium exposure on [Ca2+
. Cadmium binds to these compounds with a >1000-fold higher affinity and can produce higher fluorescence than calcium making the interpretation of this data problematic 
. A loss of a response during co-exposures of BAPTA with cadmium could be due to decreases in the effective cadmium concentrations, rather than effects on [Ca2+
. Likewise, an increase in fluorescence in fura-loaded cells following exposure to cadmium could be due to the binding of cadmium to the dye, rather than a release of intracellular calcium from storage. To circumvent this problem, a protein-based calcium indicator, yellow cameleon 3.60 was used in the current studies. In HEK293::YC3.60 cells, cadmium exposure did not elicit a change in YC3.60 fluorescence (). Under similar experimental conditions however, cadmium produced significant increases in fura-5F fluorescence ratios (). This indicates that fura-5F and potentially other BAPTA-based fluorescent dyes can be used to measure [Cd2+
, but are not appropriate when measuring the effects of cadmium on [Ca2+
To assess the effects of cadmium on intracellular calcium homeostasis, HEK293 cells that stably expressed YC3.60 were used. YC3.60 provides a direct measure of [Ca2+]i without interference from cadmium (). A second consideration in the current experimental design is the use of non-cytotoxic concentrations of metal. Exposing HEK293::YC3.60 cells to 1 µM cadmium for 4 h was sufficient to increase steady-state mRNA levels of three well-characterized cadmium-responsive genes (). In addition, exposure to 1 µM cadmium for 4 or 24 h did not produce any significant toxicological responses (). Based on these results, subsequent calcium homeostasis and signaling experiments were performed using HEK293::YC3.60 cells exposed to 1 µM cadmium. To replicate previous studies and gain an understanding of how cytotoxic levels of cadmium affect [Ca2+]i, cells were also exposed for 4 and 24 h to 30 µM cadmium, which is approximately three-times the 24 h LC50 for this cell line.
Using this experimental design, low-dose cadmium exposures did not interfere with calcium homeostasis nor deplete ER calcium store content (). Only high concentrations of cadmium (30 µM) depleted ER calcium stores. This is similar to that reported by Biagioli et al
., who observed a significant depletion of ER calcium stores in NIH 3T3 cells treated with 15 µM cadmium for 12 h 
. The reported LC50
s for cadmium in 3T3 cells range from 1–5 µM 
. These results suggest that as cells succumb to metal toxicity, calcium is released from intracellular stores.
Cadmium exposure increases the activity of MAPK and CaMK II regulated pathways 
. Since MAPKs and CaMK II are considered integrators of calcium signaling, the effect of cadmium on the expression of calcium responsive genes was investigated using cAMP/calcium signaling focused arrays. Exposure of HEK293::YC3.60 cells to non-cytotoxic levels of cadmium, 1 µM for 4 or 24 h, did not affect the expression of a significant number of genes. One gene was commonly affected by both non-cytotoxic cadmium conditions and thapsigargin-induced intracellular calcium release. Following 24 h exposure to1 µM cadmium, an additional three genes were affected by both cadmium and thapsigargin. However, among the commonly affected genes; TNF, FOS and EGR1; cadmium caused a significant decrease in their steady-state mRNA levels while an increase in [Ca2+
had the opposite effect (). These results are consistent with the lack of a significant effect on [Ca2+
in cells exposed to low concentrations of cadmium (). These metal concentrations are associated with adaptive responses and do not deplete ER calcium stores nor interfere with intracellular calcium signaling. Thus under these conditions calcium does not function as a second messenger mediating cadmium-responsive transcription.
Exposure to cytotoxic levels of cadmium affected the steady-state mRNA levels of ~60% of the genes, in contrast to non-cytotoxic conditions that affected ~2% (, and ). These results are similar to previous studies demonstrating concentration-dependent increases in the number of affected genes when cells are exposed to environmental toxicants; i.e., as the concentration of toxicant increases from adaptive to cytotoxic, there is a concomitant increase in the number of genes whose steady-state level of expression change. This was observed in HepG2 cells exposed to copper; where at physiological copper concentrations (200 µM) the expression of 30 genes was affected, but at toxicological concentrations (600 µM) the number of affected genes increased to 790 
Exposure to 30 µM cadmium for 24 h affected the expression of 50 genes. This result was consistent with the [Ca2+]i measurements in which 30 µM cadmium affected ER calcium stores (). The majority of the thapsigargin-inducible genes were also affected by 30 µM cadmium. However, three-times as many genes were affected by cadmium as thapsigargin, 17 vs. 53 (). In addition, the steady-state mRNA levels of TNF and PER1 increased in response to intracellular calcium release, but decreased following cadmium exposure. This suggests that the overlap among affected genes may be the result of a general activation of transcription by cadmium rather than a specific calcium-mediated effect.
Exposure to 30 µM cadmium caused a significant decrease in cell viability and depletion of ER calcium stores. At cytotoxic concentrations, calcium release may not be a specific cadmium-induced response; rather it could be a secondary or tertiary response, or non-specific affect. For example, cadmium exposure in rodents causes an increase in cAMP levels by increasing adenylate cyclase and decreasing cAMP phosphodiesterase activities, which ultimately leads to the activation of cAMP-dependent protein kinase regulated genes 
. Similarly, the activation of DNA damage response is due to cadmium-induced DNA damage via oxidative stress and inhibition of DNA repair, and not a direct interaction between cadmium and p53 
. In addition, the activation/suppression of transcription could be a consequence of metal-induced membrane damage and cell death. As a consequence of the breakdown of intracellular structures, calcium would be released from membrane-bound intracellular stores. Cadmium-induced oxidative stress and lipid peroxidation occur within minutes of exposure to toxic concentrations of metal and prior to any measurable cytotoxicity 
. Metal-induced damage could activate multiple processes. The activation of signaling proteins and cognate regulatory pathways would affect the expression of dozens of genes including the calcium/cAMP responsive genes on the array.
Low-level exposure to cadmium is relevant to human health as the general population is constantly exposed to low levels of this metal. Exposure to non-cytotoxic levels of cadmium is sufficient to affect gene expression, but does not alter calcium homeostasis. In addition, the transcription of calcium/cAMP responsive genes is unaffected by non-cytotoxic levels of cadmium. These data strongly suggest that cadmium-activated transcription is independent of intracellular calcium signaling. The results also support the hypothesis that at cytotoxic concentrations of cadmium, calcium-regulated signaling is affected as part of a general downstream response to cadmium-induced intracellular damage, and not a specific effect of cadmium on calcium homeostasis. They also suggest that further examination of the molecular mechanisms regulating cadmium-responsive transcription should be conducted at non-cytotoxic metal concentrations, which are environmentally relevant, and confirm that experimental reagents do not interact with cadmium.