The requirement for TRPV6 for vitamin D-dependent intestinal calcium absorption in vivo has been examined by using vitamin D-deficient TRPV6 null mice and littermate wild-type mice. Each of the vitamin D-deficient animals received each day for 4 days 50 ng of 1,25-dihydroyvitamin D 3 in 0.1 ml of 95% propylene glycol:5% ethanol vehicle or vehicle only. Both the wild-type and TRPV6 null mice responded equally well to 1,25-dihydroxyvitamin D 3 in increasing intestinal calcium absorption. These results, along with our microarray data, demonstrate that TRPV6 is not required for vitamin D-induced intestinal calcium absorption and may not carry out a significant role in this process. These and previous results using calbindin D9k null mutant mice illustrate that molecular events in the intestinal calcium absorption process in response to the active form of vitamin D remain to be defined.A primary function of vitamin D is to markedly increase intestinal absorption of calcium and phosphate (1). During the 1950s, this absorption was shown to be primarily an active calcium transport process and an independent active phosphate transport process (2, 3). With the discovery of the vitamin D endocrine system came the understanding that this process was directly stimulated by the hormonal form of vitamin D, 1␣,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) (4). Because this hormonal form of vitamin D is regulated in response to the need for calcium, it became clear that the endogenous factor discovered by Nicolaysen and Egg-Larsen is the vitamin D endocrine system producing the final vitamin D hormone, 1,25-(OH) 2 D 3 (4, 5). There is still debate whether vitamin D further influences the diffusional component of calcium absorption, taking place at high intestinal levels of calcium (6, 7).The molecular mechanism underlying active calcium transport in response to vitamin D began unraveling with the discovery of calbindin D 9k by Wasserman and colleagues (8). Schachter and colleagues (9) also found a transporter responsive to vitamin D. Others have reported that vitamin D stimulates the basal lateral membrane calcium ATPase believed to be a calcium transporter (10, 11). These components have been put together in a diagrammatic fashion to present the current hypothesis of how 1,25-(OH) 2 D 3 stimulates active intestinal calcium absorption. TRPV6 is a calcium channel protein clearly induced by 1,25-(OH) 2 D 3 (12). Calcium entering through this channel is believed to associate with calbindin D 9k , which serves as a shuttle for calcium, presenting it to the basal lateral membrane calcium ATPase. This step provides the energy input for the transfer of calcium against a concentration gradient. All three of these genes are clearly under the influence of the active form of vitamin D (13). Unfortunately not all evidence is currently in support of this hypothesis. Transgenic mice in which calbindin D 9k has been eliminated have shown that calbindin D 9k is not required for 1,25-(OH) 2 D 3 -stimulated intestinal calcium absorption (14, 15). Fo...

After a 70% partial hepatectomy (PH), the steady-state levels of Connexin (Cx)32, Cx26, and Cx43 messenger RNA (mRNA) transcripts each displayed unique patterns of temporal expression. Within 1 hour after surgical resection, increased expression of all three Cx mRNAs was observed. Subsequently, the level of Cx32 mRNA transcripts transiently decreased to a nadir at 12 hours. Comparisons of the spatial changes with previously reported hepatocyte proliferation kinetics induced by PH demonstrated that hepatocytes before S-phase "remodel" their GJs. Within 1 to 5 hours post-PH, midzonal hepatocytes exhibited diffuse membrane staining different from the normal punctate distribution. Subsequently, midzonal hepatocytes expressed colocalized punctate Cx32 and Cx26 immunostaining. Because the changes occurred in midzonal hepatocytes before 24 hours post-PH, near the peak of hepatocyte DNA synthesis, these findings indicate that Cx26 is enhanced in hepatocytes before the onset of S-phase. In contrast to the restricted expression of Cx43 in Glisson's capsule in adult liver, Cx43 protein and mRNA were enhanced specifically in proliferating bile duct and perisinusoidal cells post-PH. PH performed during continuous administration of 2-acetylaminofluorene (AAF) prevented changes in Cx32 and Cx26 staining observed in the absence of AAF. Proliferating oval cells were found to express diffuse Cx43 immunoreactivity. On day 11 post-PH and AAF, basophilic hepatocytes displayed both punctate Cx32 and Cx26 staining, whereas bile ducts and perisinusoidal cells expressed Cx43. These findings indicate that alterations in Cx32 and Cx26 expression occur rapidly in hepatocytes stimulated to proliferate and that several nonparenchymal liver cell types upregulate Cx43 expression when induced to proliferate. Differentiation of oval cells into basophilic hepatocytes resulted in their expression of Cx32 and Cx26.

Female rats were subjected to a 70% partial hepatectomy and administered either diethylnitrosamine (10 mg/kg) or the solvent, trioctanoin. After a 2 day recovery from the surgery, the rats were placed on basal diet alone or containing phenobarbital (500 mg/kg diet), mestranol (0.2 mg/kg diet), tamoxifen (250 or 500 mg/kg diet) or toremifene (250, 500 or 750 mg/kg diet) for 6 or 18 months prior to killing. The liver and kidneys were prepared for pathological diagnoses. In addition, sections of liver from the 6 month killing were frozen and serially sectioned. The sections were stained for expression of the placental isozyme of glutathione S-transferase (GST), gamma glutamyl transpeptidase (GGT), canalicular ATPase (ATP) and glucose 6-phosphatase (G6P) and scored by quantitative stereology for number and volume fraction of liver occupied by altered hepatic foci (AHF) with alterations in these markers individually and combined (ANY). Each of the agents increased the volume fraction of liver occupied by AHF when the ANY category was used. Statistical increases in both the GGT-positive and G6P-deficient AHF populations were observed in the spontaneously as well as DEN-initiated groups treated with tamoxifen or toremifene. After 18 months of administration, the highest concentration of tamoxifen increased the incidence of malignant hepatic neoplasms in non-DEN-initiated rats. Toremifene, at the highest tested dose, increased the incidence of hepatocellular carcinomas in the DEN-initiated groups to a level one-third that observed with tamoxifen administration to DEN-initiated rats. Both tamoxifen and toremifene increased the incidence of hypernephromas in previously DEN-initiated rats. While both tamoxifen and toremifene are effective promoting agents for DEN-initiated lesions, tamoxifen is more potent than toremifene in the induction of rat hepatocarcinogenesis.

Carcinogenesis develops in stages that have been operationally defined as initiation, promotion and progression. Although morphological end points have been described for detection and quantitation of these stages, to date initiation has been assessed only in the context of clonal growth in response to certain promoting agents. Initiated cells are morphologically indistinguishable from surrounding cells and early changes at the cellular level during initiation have not been clarified. One commonly used end point for the detection of preneoplastic hepatic lesions i their aberrant expression of the placental isozyme of glutathione S-transferase (PGST). Because single hepatocytes expressing PGST have been detected in aged rats and in those administered hepatocarcinogens, it has been suggested that such cells constitute a population of putatively initiated hepatocytes. In order to further elucidate the characteristics of single PGST-positive hepatocytes, we analyzed the number of these cells 2 and 18 weeks after various doses (0-100 mg/kg) of diethylnitrosamine (DEN) and of dimethylbenz[a]anthracene (DMBA). When determined 14 days after carcinogen administration, the number of single hepatocytes expressing PGST was greater after DEN administration (ranging from 0.8 +/- 0.3 per cm2 transection of liver at 1 mg/kg to 33.0 +/- 4.7 at 100 mg/kg) than after DMBA administration (ranging from 0.25 +/- 0.14 at 10 mg/kg to 3.03 +/- 0.5 at 100 mg/kg); none were detected in control rats of the same age. Additional rats were maintained on a basal diet or a basal diet plus phenobarbital for a further 4 month period. Whereas individual PGST-positive hepatocytes were only sporadically detected in rats treated with DMBA and maintained on a basal diet for 18 weeks, those rats placed on phenobarbital for 16 weeks had an even higher number of such PGST-positive hepatocytes than at 2 weeks after DMBA administration. In contrast, the dose-response curve observed for DEN-treated rats 18 weeks after carcinogen administration was similar to that observed 2 weeks after carcinogen treatment for both phenobarbital- and non-phenobarbital-treated rats. In addition, the number of single PGST-positive hepatocytes detected at 2 weeks was directly parallel to the number of altered hepatic foci expressing PGST 18 weeks after DEN administration. The dose-dependent induction of PGST-positive single hepatocytes after treatment with two hepatocarcinogens, the dose-dependent growth of altered hepatic foci (AHF) expressing PGST with phenobarbital administration and the parallel dose-response curve of single hepatocytes expressing PGST and later of AHF expressing PGST argue strongly for a precursor role of single PGST-positive cells in the development of AHF expressing PGST.

Long-term treatment of breast cancer patients with tamoxifen has prompted concern over potential toxicity of this drug with chronic administration. Since tamoxifen has estrogenic action in the rat liver and estrogenic agents can increase hepatoma incidence in rats, tamoxifen and two non-isomerizable, fixed-ring analogs (FRT1 and FRT2) were evaluated as promoting agents in a two-stage model of hepatocarcinogenesis in female Fischer F344 rats. The rats were subjected to 70% partial hepatectomy and half of the animals were administered the initiating agent, diethylnitrosamine (DEN; 10 mg/kg body wt), while the other half were not initiated. Groups of initiated and uninitiated animals were allowed to recover for 2 weeks and were then administered tamoxifen or one of the fixed-ring analogs admixed into AIN-76A diet at 25, 100 or 250 mg/kg diet. After 6 months of anti-estrogen administration the rats were sacrificed and uterine weights, blood levels of anti-estrogen, and liver histopathology were assessed. Uterine weights were decreased 2- to 3-fold by each of the agents, consistent with an anti-estrogenic action in the rat. The serum levels in rats administered 250 mg anti-estrogen/kg diet for 6 months were 320+/-20 ng/ml for tamoxifen, 320+/-10 for FRT1 and 350+/-20 for FRT2. The liver levels after a 6 month administration of 250 mg anti-estrogen/kg diet were 13 870+/-860 ng/g for tamoxifen, 13 300 +/-860 for FRT1 and 26 900+/-1900 for FRT2. A dose-dependent increase in serum and liver level of each compound was noted when measured at the 6 month time period. The number and percentage of the liver occupied by altered hepatic foci (AHF) were determined by quantitative stereology. A dose-dependent increase above initiated controls was observed in the initiated, tamoxifen-treated rats. Both fixed-ring analogs also increased the number and size of AHF compared with initiated controls, but were less potent than tamoxifen, suggesting that tamoxifen has an intrinsic promoting action in the liver that is independent of its ability to isomerize to more potent estrogenic compounds. In addition, the fixed-ring analogs have a weaker promoting activity in the rat liver than does tamoxifen. This may be due to pharmacokinetic differences at the lower two doses, but it is independent of achieved serum level at the highest dose and hence may reflect differences in intrinsic activity of these compounds. Thus tamoxifen and the two fixed-ring analogs promote the development of rat hepatocarcinogenesis.