Tumors In Female Rats Research Articles

Bisphenol A and Di(2-Ethylhexyl) Phthalate promote pulmonary carcinoma in female rats via estrogen receptor beta: In vivo and in silico analysis

The prevalence of lung cancer in women currently merits our attentions. However, cigarette exposure alone does not tell the whole story that lung cancer is more prevalent among non-smoking women. Since female lung cancer is closely linked to estrogen levels, many of endocrine disrupting chemicals (EDCs), as the substances similar to estrogen, affect hormone levels and become a potential risk of female lung cancer. Additionally, the combined toxicity of EDCs in daily environment has only been discussed on a limited scale. Consequently, this study explored the cancer-promoting effect of two representative substances of EDCs namely Bisphenol A (BPA) and Di(2-Ethylhexyl) Phthalate (DEHP) after their exposure alone or in combination, using a rat pulmonary tumor model published previously, combining bioinformatics analysis based on The Comparative Toxicogenomics Database (CTD) and The Cancer Genome Atlas (TCGA) databases. It demonstrated that BPA and DEHP enhanced the promotion of pulmonary tumor in female rats, either alone or in combination. Mechanistically, BPA and DEHP mainly directly bound and activated ESR2 protein, phosphorylated CREB protein, activated HDAC6 transcriptionally, induced the production of the proto-oncogene c-MYC, and accelerated the formation of pulmonary tumor in female rats. Remarkably, BPA, rather than DEHP, exhibited a much more critical effect in female lung cancer. Additionally, the transcription factor ESR2 was most affected in carcinogenesis, causing genetic disruption. Furthermore, the TCGA database revealed that ESR2 could enhance the promotion and progression of non-small cell lung cancer in females via activating the WNT/β-catenin pathway. Finally, our findings demonstrated that BPA and DEHP could enhance the promotion of pulmonary carcinoma via ESR2 in female rats and provide a potential and valuable insight into the causes and prevention of lung cancer in non-smoking women due to EDCs exposure.

Bisphenol A drives di(2-ethylhexyl) phthalate promoting thyroid tumorigenesis via regulating HDAC6/PTEN and c-MYC signaling

Bisphenol A (BPA) and di-(2-ethylhcxyl) phthalate (DEHP) are exist widespread in the environment and produce adverse effect to human as environmental disruptors (EDCs). Epidemiological studies have found that the exposure of DEHP and BPA could increase the susceptibility to thyroid diseases including thyroid cancer and benign thyroid nodules. Due to the existence of multiple pollutants in our daily life, the mixed toxic effects of exposure and their interrelationships may distinguish from the exposure to a single chemical, so it is of great significance to explore the mixed toxic effect of DEHP and BPA co-exposure. Thyroid, as one of the target organs of EDCs, is prone to tumor occurrence, however, whether the mixture of BPA and DEHP will affect the occurrence of thyroid cancer is still obscure. We aim to investigate the effect of single or combined exposure to BPA and DEHP on the occurrence of thyroid cancer. An animal model of exposure to BPA and DEHP was firstly established to evaluate their effect on DMD-induced thyroid cancer. Additionally, human thyroid cancer cells BCPAP and thyroid cells Nthy-ori3–1 were used to further clarify some possible mechanisms of BPA and MEHP, the main metabolite of DEHP. Consequently, we found that BPA alone could increase the incidence of thyroid tumors in female rats compared with DEHP, and DEHP enhanced the effect of BPA on cancer promotion. BPA alone and in combination with DEHP mainly induced the expression of HDAC6, inhibited tumor suppressor gene PTEN upregulated the expression of oncogene c-MYC, and eventually elevate the susceptibility to thyroid tumors. Mechanistically, BPA alone and in combination with MEHP could significantly induce the proliferation of BCPAP cells depending on HDAC6, which could modulate H3K9ac to inhibit PTEN, activate AKT signaling pathway, and simultaneously upregulate the expression of c-MYC. Interestingly, we found that BPA alone and in combination with MEHP could significantly induce the proliferation of Nthy-ori3–1 cells independent on HDAC6 via activating ERK signaling pathway. Taken together, these findings not only provide new evidence of the promoting effect of BPA and DEHP on thyroid cancer but also discusses some possible mechanisms underlying these effects.

Investigation of potential early key events and mode of action for 1,2-dichloroethane-induced mammary tumors in female rats.

1,2-dichloroethane (DCE or EDC) is a chlorinated hydrocarbon used as a chemical intermediate, including in the synthesis of polyvinyl chloride. Although DCE has induced tumors in both rats and mice, the overall weight-of-evidence suggests a lack of in vivo mutagenicity. The present study was conducted to explore a potential mode of action further for tumor formation in rat mammary tissue. Fischer 344 rats were exposed to target concentrations of 0 or 200 ppm of DCE vapors (6 hours/day, 7 days/week) for at least 28 days; 200 ppm represents a concentration of ~20% higher than that reported to induce mammary tumors. Endpoints examined included DNA damage (via Comet assay), glutathione (reduced, oxidized and conjugated), tissue DNA adducts, cell proliferation and serum prolactin levels. Exposure to DCE did not alter serum prolactin levels with consistent estrous stage, did not cause cell proliferation in mammary epithelial cells, nor result in histopathological alterations in the mammary gland. DNA adducts were identified, including the N7 -guanylethyl glutathione adduct, with higher adduct levels measured in liver (nontumorigenic target) compared with mammary tissue isolated from the same rats; no known mutagenic adducts were identified. DCE did not increase the Comet assay response in mammary epithelial cells, whereas DNA damage in the positive control (N-nitroso-N-methylurea) was significantly increased. Although the result of this study did not identify a specific mode of action for DCE-induced mammary tumors in rats, the lack of any exposure-related genotoxic responses further contributes to the weight-of-evidence suggesting that DCE is a nongenotoxic carcinogen.

Abstract 15: Hypertension drug Olmesartan medoxomil promotes colonic tumorigenesis in AOM-induced CRC rat model

Abstract Some retrospective studies suggest an inverse risk-association between the use of angiotensin signaling inhibitors and colorectal cancer (CRC). The angiotensin receptor blocker olmesartan medoxomil (OLM) is commonly prescribed for hypertension treatment. Here we sought to determine the chemopreventive efficacy of OLM using the azoxymethane (AOM)-induced CRC rat model. Male and female F344 rats (n=6/gender/group) were randomized and subcutaneously injected with 20 mg/kg of AOM once weekly for two weeks. One week later, AIN76-A diets containing OLM (0 ppm to 480 ppm) were fed to rats. Six weeks after treatment, rats were euthanized and evaluated for toxicity and aberrant crypt foci (ACF) inhibition. OLM-fed rats, particularly at ≥160 ppm, showed signs of toxicity. There was a modest decrease in the total ACFs without any change in multicrypt ACFs in male rats; however in females there was an increase in multicrypt ACFs as compared to the control diet group. For tumor efficacy evaluation, 5-week-old F344 rats were randomized (n=30/gender/group) and CRC was induced as described above. At the adenoma stage (12-weeks after AOM-treatment), rats were administered OLM (0, 20 and 40 ppm) in the diet for 33 weeks, and colonic tumors were evaluated. In the control group, there was a huge disparity in colonic tumor incidence and multiplicity between genders with ~3-fold less tumors in female rats. Surprisingly, colon tumor incidence was significantly increased (3-fold; p<0.0002) in the OLM-treated female rats (77% OLM vs 24% control). Colon tumor multiplicity analysis also showed tumor promoting effects. In the 20 ppm OLM-treated male and female rats, colonic adenocarcinoma multiplicities were increased by ~91% (p<0.01) and ~362% (p<0.0001), respectively, when compared with their respective controls. Although there was no dose response, similar tumor promoting effects were found in 40 ppm OLM-treated rats. To understand male vs female colon tumor disparities and OLM tumor promoting effects, we carried out RNA-Seq analysis of colonic tumors. Reflecting tumor data, gene expression analysis also showed significant differences between male and female rats with respect to type II-interferon, PI3K-AKT, cholesterol, lipid droplet, mucin, IL-3, cell-cycle and nucleotide GPCR signaling pathways. OLM treatment significantly upregulated 586 and 740 genes, and downregulated 648 and 564 genes in male and female rat tumors, respectively. Of the 293 common genes identified, Irx5, Lrrn1, Ly6g6e, Sccpdh, Hoxd12, Igfbp1, Krt81 and DSC3 were upregulated up to 245-fold; and Rfxapl1, Serpinb3a, REg3b, Bnc1 and Ak4 were down-regulated 10 - 200-fold with OLM treatment. In conclusion, chronic administration of OLM did not provide any chemopreventive benefit against colonic tumors in the AOM-induced F344 rat CRC model. Further studies are warranted to assess the risk of colon cancer associated with long-term OLM use, particularly in females. (Supported by NCI HHSN 261201500038I and VA Merit Award) Citation Format: Venkateshwar Madka, Yuting Zhang, Gopal Pathuri, Janani Panneerselvam, Nicole Stratton, Anil Singh, Shizuko Sei, Jennifer Fox, Chinthalapally V. Rao. Hypertension drug Olmesartan medoxomil promotes colonic tumorigenesis in AOM-induced CRC rat model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 15.

Maternal western-style diet enhances the effects of chemically-induced mammary tumors in female rat offspring through transcriptome changes

Previous studies have shown that early life intake of high-fat diet or western-style diet (WD) enhances the development of mammary tumors in adult female rats. Thus, we hypothesized that maternal WD throughout pregnancy and the lactation period could speed up the development of MNU-induced mammary tumors and alter their gene expression. For this, the present study investigated the gene expression profile of chemically-induced mammary tumors in female rat offspring from dams fed a WD or a control diet. Pregnant female Sprague-Dawley rats received a WD (high-fat, low-fiber and oligoelements) or a control diet from gestational day 12 until post-natal day (PND) 21. At PND 21, female offspring received a single dose of N-Methyl-N-Nitrosourea (MNU, 50 mg/kg body weight) and were fed a control diet for 13 weeks. Tumor incidence, multiplicity, and latency were recorded and mammary gland samples were collected for histopathology and gene expression analysis. Tumor multiplicity and histological grade were significantly higher and tumor latency was lower in WD offspring compared to control offspring. Transcriptome profiling identified 57 differentially expressed genes in tumors from WD offspring as compared to control offspring. There was also an increase in mRNA expression of genes such as Emp3, Ccl7, Ets1, Abcc5, and Cyr61, indicative of more aggressive disease detected in tumors from WD offspring. Thus, maternal WD diet increased MNU-induced mammary carcinogenesis in adult female offspring through transcriptome changes that resulted in a more aggressive disease.

Investigation of the potential non-genotoxic mode of action for 1,2-Dichloroethane (DCE)-induced mammary tumors in female rats

Investigation of the potential non-genotoxic mode of action for 1,2-Dichloroethane (DCE)-induced mammary tumors in female rats

Carcinogenic activity of pentabrominated diphenyl ether mixture (DE-71) in rats and mice.

Pentabrominated diphenyl ether (PBDE) flame retardants have been phased out in Europe and in the United States, but these lipid soluble chemicals persist in the environment and are found human and animal tissues. PBDEs have limited genotoxic activity. However, in a 2-year cancer study of a PBDE mixture (DE-71) (0, 3, 15, or 50 mg/kg (rats); 0, 3, 30, or 100 mg/kg (mice)) there were treatment-related liver tumors in male and female Wistar Han rats [Crl:WI(Han) after in utero/postnatal/adult exposure, and in male and female B6C3F1 mice, after adult exposure. In addition, there was evidence for a treatment-related carcinogenic effect in the thyroid and pituitary gland tumor in male rats, and in the uterus (stromal polyps/stromal sarcomas) in female rats. The treatment-related liver tumors in female rats were unrelated to the AhR genotype status, and occurred in animals with wild, mutant, or heterozygous Ah receptor. The liver tumors in rats and mice had treatment-related Hras and Ctnnb mutations, respectively. The PBDE carcinogenic activity could be related to oxidative damage, disruption of hormone homeostasis, and molecular and epigenetic changes in target tissue. Further work is needed to compare the PBDE toxic effects in rodents and humans.

Prognostic factors in MNU and DMBA-induced mammary tumors in female rats

Chemically-induced mammary tumors in rats by the carcinogens 1-methyl-1-nitrosourea- (MNU) and 7,12-dimethylbenz[a]anthracene (DMBA) are the most widely used models for studies related with human breast cancer. This study aimed to evaluate the immunoexpression of the prognostic factors estrogen receptor α (ERα), progesterone receptor (PR) and Ki-67, in MNU and DMBA-induced rat mammary tumors, in order to know the model that best suits to woman breast cancer. Twenty-eight MNU-induced and 16 DMBA-induced mammary tumors in virgin female Sprague-Dawley rats were analyzed. The expression of the prognostic markers ERα, PR and Ki-67 proliferation index (Ki-67 PI) was assessed by immunohistochemistry. Mitotic activity index (MAI) was also evaluated. More than one histological pattern was identified in each mammary tumor. Carcinomas constituted the lesions most frequently induced by both carcinogens: 33 MNU-induced carcinomas and 23 DMBA-induced carcinomas. All MNU and DMBA-induced mammary carcinomas were ER+/PR+, with a higher expression of ERα when compared with PR. Tumors’ weight, the expression of ERα, PR, Ki-67 PI and MAI were higher in MNU-induced mammary carcinomas when compared with the DMBA-induced ones. Statistically significant differences between groups were observed for ERα, PR and MAI (p<0.05). The higher KI-67 PI and MAI in MNU-induced mammary carcinomas are suggestive of a higher aggressiveness of these carcinomas when compared with the DMBA-induced ones, and consequently a worse response to the therapy and a worse prognosis. In this way, the use of the rat model of MNU-induced mammary tumors is advised in experimental protocols aiming to study more aggressive mammary tumors within the group of double-positive mammary tumors (ER+/PR+).

Flometoquin (Pesticides).

Food Safety Commission of Japan (FSCJ) conducted a risk assessment of flometoquin (CAS No. 875775-74-9), a quinoline insecticide, based on results from various studies. Major adverse effects of flometoquin observed were suppressed body weight and hepatocellular steatosis in rats, and ovarian atrophy with decreased numbers of small follicle in rats and mice. Neither teratogenicity nor genotoxicity relevant to human health was detected. Increased incidences of gonadal stromal tumor in female rats and of small intestine adenocarcinomas in male mice were identified in carcinogenicity studies. Genotoxic mechanisms were, however, unlikely involved in their tumor developments, and these enabled FSCJ to establish a threshold in the assessment. Mechanism and toxicity studies suggested that ovarian atrophy triggered the development of gonadal stromal tumor, through continuous stimulation of gonadotropin to the gonadal stroma, via negative feedback. A reproductive study showed the decreases in numbers of small follicle, implantation and also in litter size. Based on the results from various studies, flometoquin (parent compound only) was the residue definition for dietary risk assessment in agricultural products. The lowest no-observed-adverse-effect level (NOAEL) obtained from all the studies was 0.8 mg/kg bw/day in a developmental toxicity study in rabbits. FSCJ specified an acceptable daily intake (ADI) of 0.008 mg/kg bw/day by applying a safety factor of 100 to the NOAEL. FSCJ recognized that in considering the ambiguity of the underlying mechanism, the adverse effect on small follicle possibly occurred after single oral administration of flometoquin. Thus FSCJ specified an acute reference dose (ARfD) to be 0.044 mg/kg bw by applying a safety factor of 100 to the NOAEL of 4.45 mg/kg bw per day in a two-generation reproductive toxicity study in rats, based on a comprehensive evaluation of NOAEL for ovarian toxicity.

Electron Microscopy Findings in N-Methyl-N-Nitrosourea-Induced Mammary Tumors.

Although the rat model of mammary tumors chemically induced by N-methyl-N-nitrosourea (MNU) has been frequently used by several research teams, there is a lack of ultrastructural studies in this field. The main aim of this work was to perform an ultrastructural characterization of MNU-induced mammary tumors in female rats. Some alterations previously reported in human mammary tumors, such as nucleus size and shape, accumulation of heterochromatin in the perinuclear region, and interdigitating cytoplasmic processes between cancer cells were also observed in MNU-induced mammary tumors. Although a low number of samples were analyzed by transmission electron microscopy in the present study, we consider that it may contribute to a better understanding of MNU-induced mammary carcinogenesis in a rat model. The ultrastructural characteristics of the two most frequently diagnosed mammary carcinomas described in the present work can be useful to differentiate them from other histological patterns. In addition, the loss of cytoplasm in neoplastic cells and formation of vacuoles were described.

Development of an inhalation unit risk factor for ethylene dichloride

The Texas Commission on Environmental Quality (TCEQ) conducts up-to-date carcinogenic assessments for chemicals emitted in Texas. An inhalation unit risk factor (URF) was developed for ethylene dichloride (EDC, CAS 107-06-2) based on tumorigenicity results observed in a 2-year animal inhalation study conducted by Nagano et al. More specifically, the incidence of combined mammary gland tumors (adenomas, fibroadenomas, adenocarcinomas) in female rats demonstrated a statistically significant dose–response relationship, was amenable to benchmark concentration (BMC) modeling, was ultimately determined to be the most sensitive tumorigenic effect in the most sensitive species and sex, and was utilized as the carcinogenic endpoint for the development of the URF. The 95% lower confidence limit of the BMC at the 10% excess risk level (BMCL10 of 40.1 ppm) was determined for calculation of the URF. The resulting URF based on increased incidence of combined mammary gland tumors in female rats is 1.4E-02 per ppm (3.4E-03 per mg/m3). The lifetime air concentration corresponding to a no significant excess risk level of 1 in 100 000 is 0.71 ppb (2.9 μg/m3), which is considered sufficiently health-protective for use in protecting the general public against the potential carcinogenic effects of chronic exposure to EDC in ambient air.

Crystal structure and chemotherapeutic efficacy of the novel compound, gallium tetrachloride betaine, against breast cancer using nanotechnology.

The objective of this study was to investigate the antitumor efficacy of a novel synthesized compound, betaine gallium-tetrachloride (BTG), alone or combined with ZnO-nanoparticles (BTG + ZnO-NPs) on the incidence of 7, 12-dimethylbenz-anthrathene-induced mammary tumor in female rats. Crystal and molecular structure of the prepared BTG were identified using X-ray crystallography. In vitro study revealed BTG more cytotoxic than BTG + ZnO-NPs on human breast cancer (MCF-7) cell line. In vivo study demonstrated that the blood antioxidant status of tumor-bearing rats (DMBA group) was significantly lower than normal noticeable by a significant decrease in GSH content, GPx, SOD, and CAT activities associated with a significantly high MDA content. Both treatments have significantly elevated SOD and CAT activities with a concomitant decrease of MDA level compared to DMBA group. However, BTG + ZnO-NPs accentuated the decrease of GSH regarding DMBA group. The results showed also that both treatments significantly activate caspase-3 enzyme and apoptosis in mammary glands. Their administration to tumor-bearing rats was found to significantly reduce plasma iron and iron-binding capacity (TIBC) compared to DMBA group. Regarding liver function, both treatments significantly reduced the increase of ALT and AST activities compared to DMBA group. However, BTG + ZnO-NPs decreased albumin below normal level. Histopathological studies showed that normalization of tissue structures was higher in BTG than BTG + ZnO-NPs treatment. According to the results obtained, it is observed that the antitumor effect of BTG alone was as strong as BTG + ZnO-NPs and even more efficient in some aspects accordingly, a combination is not needed. Thus, the novel synthetic gallium derivatives may potentially present a new hope for the development of breast cancer therapeutics, which should attract further scientific and pharmaceutical interest.

Combination between Taxol-Encapsulated Liposomes and Eruca sativa Seed Extract Suppresses Mammary Tumors in Female Rats Induced by 7,12 Dimethylbenz(α)anthracene.

Taxol (paclitaxel) is a powerful anti-cancer drug widely used against several types of malignant tumors. Because Taxol may exert several side effects, a variety of formulations have been developed. One of these features liposomes, regarded as one of the most promising drug carriers, biocompatible and best able to reduce drug toxicity without changing efficacy against tumor cells. Eruca sativa seed extract (SE) is considered a promising natural product from cruciferous vegetables against breast cancer, increasing chemotherapeutic and eliminating harmful side effects. The effects of Taxol-encapsulated liposomes (T) alone and in combination between Eruca sativa seed extract on nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2) and B-cell lymphoma-2 (Bcl-2) gene expression levels were investigated in rat mammary gland carcinogenesis induced by 7,12 dimethylbenz(α) anthracene (DMBA) using qRT-PCR. The results showed that DMBA increased NF-κB, COX-2 and Bcl-2 gene expression levels and lipid peroxidation (LP), while decreasing glutathione-S-transferase (GST) and superoxide dismutase (SOD) activities and total antioxidant concentration (TAC) compared to the control group. T and T-SE treatment reduced NF-κB, COX-2 and Bcl-2 gene expression levels and LP. Hence, T and T-SE treatment appeared to reduce inflammation and cell proliferation, while increasing apoptosis, GST and SOD activities and TAC.

A high dose mode of action for tetrabromobisphenol A-induced uterine adenocarcinomas in Wistar Han rats: A critical evaluation of key events in an adverse outcome pathway framework

TBBPA is a non-genotoxic flame retardant used to improve fire safety in a wide variety of consumer products. Estimated human exposures to TBBPA are very low (<0.000084 mg/kg-day), relative to the doses (500 and 1000 mg/kg-day of TBBPA) administered in a recent bioassay that resulted in uterine tumors in Wistar Han rats following chronic exposure. As part of an effort to characterize the relevance of the uterine tumors to humans, data and biological knowledge relevant to the progression of events associated with TBBPA-induced uterine tumors in female rats were organized in an adverse outcome pathway (AOP) framework. Based on a review of possible MOAs for chemically induced uterine tumors and available TBBPA data sets, a plausible molecular initiating event (MIE) was the ability of TBBPA to bind to and inhibit estrogen sulfotransferases, the enzymes responsible for sulfation of estradiol. Subsequent key events in the AOP, including increased bioavailability of unconjugated estrogens in uterine tissue, would occur as a result of decreased sulfation, leading to a disruption in estrogen homeostasis, increased expression of estrogen responsive genes, cell proliferation, and hyperplasia. Available data support subsequent key events, including generation of reactive quinones from the metabolism of estrogens, followed by DNA damage that could contribute to the development of uterine tumors. Uncertainties associated with human relevance are highlighted by potential strain/species sensitivities to development of uterine tumors, as well as the characterization of a dose-dependent MIE. For the latter, it was determined that the TBBPA metabolic profile is altered at high doses (such as those used in the cancer bioassay), and thus an MIE that is only operative under repeated high dose, administration. The MIE and subsequent key events for the development of TBBPA-induced uterine tumors are not feasible in humans given differences in the kinetic and dynamic factors associated with high dose exposures in rats relative to human exposure levels to TBBPA.

Dose- and time-dependent changes in tissue levels of tetrabromobisphenol A (TBBPA) and its sulfate and glucuronide conjugates following repeated administration to female Wistar Han Rats.

Tetrabromobisphenol A (TBBPA), a nongenotoxic flame retardant, causes uterine tumors in female rats. A proposed mode of action (MoA) for these tumors involves an increase in the bioavailability of estradiol as a result of TBBPA inhibiting estrogen sulfotransferases (ES), the enzymes responsible for inactivating and enhancing the elimination of estradiol. The objective of this study was to evaluate the effect of dose and repeated administration of TBBPA on the level of TBBPA, TBBPA-glucuronide (GA) and TBBPA-sulfate (S) conjugates in plasma, liver and uterus of female Wistar Han rats administered TBBPA (50, 100, 250, 500 or 1000 mg/kg) for 28 consecutive days. In accordance with this objective, TBBPA sulfation was used as a surrogate for evaluating the potential for estradiol sulfation to be limited at high dose levels of TBBPA. Blood samples were collected at 4 and 8 h post-dosing on study day 7, 14, and 28, while liver and uterus were collected at the same time points following 28 days of dosing. Tissue samples were analyzed for TBBPA, TBBPA-GA and TBBPA-S by LC–MS/MS. A dose-related increase in the concentration of all three analytes occurred in plasma (day 7, 14, and 28) as well as liver and uterus tissue (day 28) at both 4 and 8 h post dose. The plasma concentration of TBBPA-GA and TBBPA-S was higher in animals dosed for 28 days compared to those dosed for 7 or 14 days showing an increase in systemic circulation of these conjugates with repeated administration. The balance of these conjugates was also different in tissues with TBBPA-S > TBBPA-GA at high doses in the liver and TBBPA-GA > TBBPA-S in both plasma and uterus. In all three tissues the ratio of TBBPA-S/TBBPA-GA showed a decreasing trend with dose, suggesting that at high TBBPA dose levels sulfation of TBBPA becomes limited. This effect was most apparent in the liver and plasma at 28 days of administration. Together these data show that administration of high doses of TBBPA associated with the induction of uterine tumors, results in a disruption in the balance of conjugates reflected by a decrease in the TBBPA-S/TBBPA-GA ratio. A limitation in the sulfation of TBBPA in vivo supports in vitro data defining TBBPA as an inhibitor of ES activity, thus providing further support that the proposed MoA occurs under conditions of high dose, chronic TBBPA administration to Wistar Han rats. Given that the uterine tumors observed in rats (250–1000 mg/kg-day) only occur at very high doses that perturb homeostatic control, it is unlikely such effects would occur in humans given that current TBBPA exposure levels are approximately eight orders of magnitude lower than these doses that are associated with exceeding the capacity of conjugation pathways in animal studies.

Whey Protein Hydrolysate but not Whole Whey Protein Protects Against 7,12-Dimethylbenz(a)anthracene-Induced Mammary Tumors in Rats

Effects of intact and processed bovine milk proteins on development of chemically induced mammary tumors in female rats were compared. AIN-93G diets were made with 20% casein (CAS), casein hydrolysate (CASH), intact whey protein (IWP), or whey protein hydrolysate (WPH). Pregnant Sprague-Dawley rats were fed the diets starting at Gestational Day 4. Offspring were fed the same diet. At 50 days, female offspring (44–49/group) were gavaged with sesame oil containing 80 mg/kg of the mammary carcinogen dimethylbenzanthracene (DMBA) and euthanized 62 days posttreatment. Rats fed WPH had an adenocarcinoma incidence of 17% compared to the rats fed CAS, CASH, and IWP diets (34%, 33%, and 36% respectively) (P < 0.001). Median palpable tumor latency for rats fed WPH was greater (61 days, P < 0.001) compared to CAS (44 days), CASH (42 days) and IWP (45 days). Tumor multiplicity was also lower (1.5 vs. 3.0, P < 0.05) in rats fed WPH than in CAS and CASH fed groups. Results demonstrate that hydrolytic processing of whey protein is required for this diet to be effective in reducing DMBA-induced mammary tumors. The bioactive compounds produced during whey protein processing and mechanisms underlying the anticancer effects of WPH are yet to be identified.

One Year Exposure to Nocturnal 7 Hz, Amplitude-Modulated Magnetic Fields Suppresses Clinical Expression of DMBA Induced Tumors in Female Rats

Female rats that had received only four oral administrations of Dimethylbenz(a) anthracene (DMBA) were exposed for one year every night for about 6 min every hour between midnight and 08 hr to various intensities of 7 Hz, amplitude modulated magnetic fields generated through Helmholtz coils. The rats exposed to intensities between 400 and 500 nT did not develop any overt tumors even though they received DMBA. On the other hand rats exposed to the intensities between 30 and 60 nT developed a variety of different, qualitatively unusual tumors that were located within pancreatic, salivatory, and nasal tissues. Their histological features are presented. These results should be considered preliminary but suggest that protracted exposures to particularly patterned and intensity magnetic fields during the nocturnal cycle may suppress the chemical reactions that contribute to the nuclear changes in the cell or the intercellular cohesive networks that ultimately trigger these massive proliferations of tissue.

Treatment with Insulin Analog X10 and IGF-1 Increases Growth of Colon Cancer Allografts

Obesity and type 2 diabetes are associated with an increased risk for development of certain forms of cancer, including colon cancer. The publication of highly controversial epidemiological studies in 2009 raised the possibility that use of the insulin analog glargine increases this risk further. However, it is not clear how mitogenic effects of insulin and insulin analogs measured in vitro correlate with tumor growth-promoting effects in vivo. The aim of this study was to examine possible growth-promoting effects of native human insulin, insulin X10 and IGF-1, which are considered positive controls in vitro, in a short-term animal model of an obesity- and diabetes-relevant cancer. We characterized insulin and IGF-1 receptor expression and the response to treatment with insulin, X10 and IGF-1 in the murine colon cancer cell line (MC38 cells) in vitro and in vivo. Furthermore, we examined pharmacokinetics and pharmacodynamics and monitored growth of MC38 cell allografts in mice with diet-induced obesity treated with human insulin, X10 and IGF-1. Treatment with X10 and IGF-1 significantly increased growth of MC38 cell allografts in mice with diet-induced obesity and we can therefore conclude that supra-pharmacological doses of the insulin analog X10, which is super-mitogenic in vitro and increased the incidence of mammary tumors in female rats in a 12-month toxicity study, also increase growth of tumor allografts in a short-term animal model.

Carcinogenicity and hormone studies with the tissue‐selective estrogen receptor modulator bazadoxifene

Bazedoxifene Acetate (BZA) is a selective estrogen receptor modulator (SERM) that is approved for the prevention and/or treatment of osteoporosis in postmenopausal women. To assess for carcinogenic potential, BZA was administered ad libitum in the diet to rats for 2 years. BZA caused an increase in benign ovarian tumors in female rats and decreased incidences of mammary tumors (females) and pituitary tumors (males and females). In addition, BZA provided a significant survival benefit at all dosages tested, which correlated with a significant reduction in pituitary and mammary gland tumors and decreased body weight gain (both genders). Additional studies were subsequently conducted in rats and monkeys to further explore the mechanisms likely responsible for the observed effects. Results from studies in hypophysectomized and chemically castrated female rats indicated that BZA did not directly stimulate formation of ovarian cysts, but an intact pituitary was required for cyst formation. Further, BZA increased estradiol concentrations in rats and monkeys. In monkeys, BZA increased concentrations of luteinizing hormone (LH) after onset of treatment and prohibited the preovulatory surge of LH until after cessation of treatment. These hormonal changes suggest that BZA inhibited both the positive and negative feedback effects of estrogen on gonadotropins and the resulting increase in LH caused formation and persistence of ovarian cysts, which eventually transformed into benign ovarian granulosa cell tumors in the rat carcinogenicity study. These results also suggest that the reductions in pituitary and mammary gland tumors were attributed to BZA-related antagonism of endogenous estrogens at the estrogen receptors.

Event‐weighted proportional hazards modelling for recurrent gap time data

The analysis of gap times in recurrent events requires an adjustment to standard marginal models. One can perform this adjustment with a modified within-cluster resampling technique; however, this method is computationally intensive. In this paper, we describe a simple adjustment to the standard Cox proportional hazards model analysis that mimics the intent of within-cluster resampling and results in similar parameter estimates. This method essentially weights the partial likelihood contributions by the inverse of the number of gap times observed within the individual while assuming a working independence correlation matrix. We provide an example involving recurrent mammary tumours in female rats to illustrate the methods considered in this paper.