Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.

Nano-sized titanium dioxide (TiO 2 ) particles are used in sunscreen formulations to protect against skin lesions caused by exposure to UV light (Gelis et al., 2003;Rouabhia et al., 2002;Suzuki, 1987). Nano and larger scale titanium dioxide particles are known to be carcinogenic to the rat lung (Baan et al., 2006;Baan, 2007). Recently, we demonstrated a promoting effect on rat lung carcinogenesis by nano-size TiO 2 particles administered by a novel intrapulmonary spraying method (Xu et al., 2010). The mechanism of promotion of lung carcinogenesis involved the induction of MIP1α protein expression by ncTiO 2 -laden alveolar macrophages (Xu et al., 2010).We also examined the carcinogenic effect of TiO 2 (mean manufacturer's particulate diameter of 20 nm) on the skin in a UVB-initiated two-stage rat carcinogenesis model and found that topical application of TiO 2 did not promote skin carcinogenesis in this model. This result is probably due to the inability of TiO 2 to penetrate through Lack of promoting effect of titanium dioxide particles on chemically-induced skin carcinogenesis in rats and mice ABSTRACT -Nano-sized titanium dioxide particles (TiO 2 ) are widely used in cosmetics, sunscreens and food additives. We previously reported that topical application of non-coated rutile type TiO 2 did not exhibit a promoting effect on ultraviolet B-initiated skin carcinogenesis in rats, and that this was likely due to lack of penetration of TiO 2 into the epidermis. In the present study, we examined the promoting effect of silicone coated TiO 2 (sTiO 2 ) suspended in silicone oil and non-coated TiO 2 (ncTiO 2 ) suspended in Pentalan 408 on a two-stage skin chemical carcinogenesis model: sTiO 2 suspended in silicon oil forms smaller particles than ncTiO 2 suspended in Pentalan because of the smaller sizes of aggregates formed. The model used skin carcinogenesis-sensitive human c-Ha-ras proto-oncogene transgenic mice (rasH2) and rats (Hras128) and their wild-type counterparts and CD-1 mice to test the effects of topical application of TiO 2 . Animals were initially treated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) and then with 0, 10, or 20 mg sTiO 2 (mice) or 0, 50, or 100 mg ncTiO 2 (rats). The incidence and multiplicity of skin tumors (squamous cell papilloma and carcinoma) did not increase over DMBA alone controls in skin carcinogenesis-sensitive mice or rats or wild-type animals. Analysis of rat skin indicated that sTiO 2 and ncTiO 2 did not penetrate though either healthy or damaged skin. Furthermore sTiO 2 did not penetrate an in vitro human epidermis model. Our results indicate that treatment with sTiO 2 or ncTiO 2 did not promote skin carcinogenesis in mice or rats, probably due to lack of penetration through the epidermis.

The recent widespread application of Cre/loxP technology has resulted in a new generation of conditional animal models that can better recapitulate many salient features of human disease. These models benefit from the ability to monitor the expression and functionality of Cre protein. We have generated a conditional (Cre/loxP dependent) LacZ reporter rat (termed the LacZ541 rat) to monitor Cre in transgenic rats. When LacZ541 rats were bred with another transgenic rat line expressing Cre recombinase under the control of the CAG promoter, LacZ/Cre double transgenic embryos displayed ubiquitous expression of LacZ, and when LacZ541 rats were bred with transgenic rats expressing Cre/loxP-dependent oncogenic H- or K-ras, LacZ was expressed in the lesions resulting from the activation of the oncogene. The LacZ541 rat enables evaluation of the performance of Cre-expressing systems which are based upon transgenic rats or somatic gene transfer vectors and provides efficient and simple lineage marking.

Tribbles-related protein 3 (TRB3) has been shown to be a crucial modulator of tumorigenesis. However, the precise role and the functional morphology of TRB3 are not clearly understood. To elucidate these enigmas we established the cell line, M2TRB3, by introducing the human TRB3 gene and protein in Cl66M2 (M2) mouse mammary tumor cells. This cell line stably expressed the TRB3 gene and protein. After 72 h of cell culture, there was a 34% increase in the growth of M2TRB3 cells compared to the control M2 mock cells. The mean volume of the tumors originating from the M2TRB3 cells was significantly increased by 38% when compared to the mean volume of the M2 mock tumors, and the proliferating cell nuclear antigen (PCNA) labeling index in the M2TRB3 tumors was higher when compared to that of the M2 and M2 mock cells. In the tumor tissue samples, the mean diameter of nuclei in the M2TRB3 tumor cells (9.4±0.3 µm) showed a significant increase compared to that of the M2 mock tumor cells (7.0±0.2 µm). M2TRB3 cells also showed a marked increase in the population of tetraploid or octaploid nuclei compared to M2 mock cells bearing mainly either diploid or tetraploid nuclei. Western blot analysis revealed the overexpression of cyclin B1 and cyclin D1 in M2TRB3 cells when compared to that in the M2 mock cells. These novel findings provide further evidence that TRB3 promotes cell proliferation and chromosomal instability by causing polyploidization during development.