Several recent reviews have evaluated potential modes of action by which arsenic could act as a carcinogen (Kitchin, 2001
; Rossman, 2003
; Kligerman and Tennant, 2006). The goal of the present study was to evaluate the effect of life-stage on the incidence of arsenic-induced proliferative lesions and neoplasia in C3H mice. There is experimental evidence that early-life exposures (i.e. transplacental, neonatal, and pre-pubescent) to carcinogens with a mutagenic or an estrogenic mode of action may increase the incidence and multiplicity of tumor incidence compared to adult only exposures (Newbold et al., 1990
and Rice, 1976
). Previous transplacental exposure studies have demonstrated an increase in arsenic-induced proliferative lesions and neoplasia in C3H and CD-1 mice (Waalkes, et al., 2003
). To further examine the impact of age of exposure on arsenic-induced tumorigenesis we report the comparative results of long, chronic arsenic exposure beginning at in utero
, pre-pubescent and post-pubescent ages in C3H mice.
Similar to a previous report (Waalkes, et al., 2006
), we found that arsenic increased the incidence of urogenital proliferative lesions in female mice exposed in utero
. However, in our one-year exposure study the increased incidence was only observed in mice that received arsenic chronically following the in utero
or pre-pubescent exposure. The lack of a statistical increase in the in utero
only exposed group may, in part, be due to the reduced one-year observation period. A one-year exposure period was used in this study because of the high background rate of hepatocellular tumors and mortality observed in this mouse strain after one year of age. Nevertheless, chronic arsenic exposure increased the incidence of urogenital proliferative lesions (urinary bladder, uterine and oviduct hyperplasia) as compared to the control group and the in utero
only treatment group (p≤0.01 and p<0.001, respectively). This suggests that long term exposure to arsenic that includes in utero
or pre-pubescent exposures can increase the risk for urogenital effects. This is also consistent with a previous study (Waalkes et al., 2006
) in which arsenic administered in utero
, and followed by neonatal exposure to the synthetic estrogen diethylstilbestrol (DES), resulted in increased urogenital proliferative lesions and neoplasia. Both our study and the Waalkes et al. 2006
study demonstrated more than double the incidence in urinary bladder lesions with subsequent arsenic or DES exposures after the initial in utero
arsenic exposure. The increased incidence of urinary bladder hyperplasia reported here is also consistent with a previous epidemiology study done in Chile where human populations exposed to arsenic early in life through adulthood had a greater urinary bladder cancer mortality rate (Marshall et al., 2007
) than those exposed as adults only.
Unlike previous reports with in utero
arsenic exposure alone (Waalkes et al., 2003
) the incidence of liver and adrenal adenomas was dramatically reduced in mice continuously exposed to arsenic following an in utero
exposure. This apparent inhibition of tumor development was unexpected and may have been associated with altered fatty acid/cholesterol levels, reduced weight gain, and/or apoptosis of preneoplastic liver cells. Male mice kept on arsenic after the in utero
exposure weighed 20-25% less than control mice beginning on postnatal day 32 through postnatal day 365. However, food consumption was not significantly different between these groups during the course of the one-year study. Our companion study (in preparation) investigating hepatic gene expression patterns in these mice demonstrated a marked reduction in stearoyl-CoA desaturase-1 (Scd1
) mRNA in mice continuously exposed to arsenic for one year compared to controls and the in utero
only treatment group 2. Scd1
is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids and its down regulation is associated with reduced adiposity in mice (Ntambi et al., 2002
). In addition to reduced weight gain, lower mRNA transcript levels of this gene are also associated with a reduction in spontaneous liver tumor formation (Ntambi et al., 2002
and Falvella et al., 2002
). Serum cholesterol levels also influence hepatic Scd
mRNA expression in mice (Ntambi 1999
). Total cholesterol levels were lowered in male mice by more that 35% in our study and may account, in part, for the reduction in Scd
mRNA and concomitant reduction in weight gain and lack of tumor response in male mice.
Increased apoptosis may also be involved in decreased liver tumor development observed in male mice chronically exposed to arsenic. In a previous mouse study (Liu et al., 2006
), sodium arsenite at doses at or exceeding 1 mg/kg significantly reduced hepatocellular carcinoma cell growth after twenty days of i.p. treatment. However, doses below 1 mg/kg increased liver tumor growth. It is possible that a one year exposure to a lower concentration of arsenic (i.e. 5-10 ppm) in our study may not have caused such a dramatic decrease in liver tumor incidence, and perhaps may have caused an increase in liver tumor incidence in C3H mice. When apoptosis was assessed in these mouse tumors immunohistochemically, a dose-dependent increase in terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) was observed at doses of 1 mg/kg and higher, suggestive of an increased apoptotic frequency. Because doses used in our study were of a similar magnitude (~8 mg/kg) with an even longer duration compared to this previous report, a similar mechanism of increased apoptosis may be involved in reduction in liver tumor incidence observed in our study. Furthermore, our companion study (in preparation) investigating hepatic gene expression patterns in these mice demonstrated significant differences in apoptotic transcript levels in chronically exposed mice vs in utero
only exposed or control mice. In summary, these one-year observational studies describe the impact of early life-stage and prolonged arsenic exposure on arsenic-induced proliferative lesions and neoplasia in mice. Continuous exposure to inorganic arsenic, extending from gestation through one year, increases the incidence and severity of urogenital proliferative lesions in female mice and decreases the incidence of liver and adrenal tumors in male mice. The paradoxical nature of these effects may be related to altered lipid metabolism, the effective dose in each target organ and/or the shorter one-year observational period for tumor incidence. Although drinking water exposures to inorganic arsenic may enable high levels of inorganic arsenic to reach the liver, higher levels of organic forms like dimethylarsinic acid (DMA(V)) may be important in urinary bladder toxicity and hyperplasia. Previous rat studies by multiple laboratories have demonstrated that dietary or drinking water administration of DMA(V) causes urinary bladder tumors following long term exposure (Wei et al., 1999
, Arnold et al., 2006
). In contrast, high levels of sodium arsenite in the mouse liver may facilitate increased apoptosis of preneoplastic hepatocytes. Additional laboratory studies will need to be performed to substantiate these conclusions.
These studies provide additional evidence of differential age susceptibility to arsenic carcinogenesis. The duration of arsenic exposure was the major contributor to the differential tumor induction observed in male and female mice. Based on these findings, dose and length of exposure can significantly alter the incidence and severity of proliferative lesions and neoplasia following brief early-life arsenic exposures.