Systematic review of the potential carcinogenicity of bisphenol A in humans.

Bisphenol A (BPA) is a synthetic chemical to which humans are exposed through a variety of environmental sources. We have conducted a comprehensive, systematic review of 29 epidemiology studies and 27 experimental animal studies, published through May 2022, evaluating the potential carcinogenicity of BPA to contribute to the understanding of whether BPA is carcinogenic in humans. We conducted this review according to best practices for systematic reviews and incorporating established frameworks for study quality evaluation and evidence integration. The epidemiology studies have many limitations that increase the risk of biased results, but overall, the studies do not provide clear and consistent evidence for an association between BPA exposure and the development of any type of cancer. The experimental animal studies also do not provide strong and consistent evidence that BPA is associated with the induction of any malignant tumor type. Some of the proposed mechanisms for BPA carcinogenicity are biologically plausible, but the relevance to human exposures is not clear. We conclude that there is inadequate evidence to support a causal relationship between BPA exposure and human carcinogenicity, based on inadequate evidence in humans, as well as evidence from experimental animal studies that suggests a causal relationship is not likely.

Derivation of an oral Maximum Allowable Dose Level for Bisphenol A.

Bisphenol A (BPA) is a high production volume chemical that is used in plastics and epoxy coatings. In 2015, California's Office of Environmental Health Hazard Assessment (OEHHA) added BPA to the Proposition 65 list of chemicals "known to cause reproductive toxicity" based on its Developmental and Reproductive Toxicant Identification Committee's (DART-IC) conclusion that BPA has been shown to cause female reproductive toxicity. A critical factor in determining compliance with Proposition 65 is a Maximum Allowable Dose Level (MADL), which is the exposure level at which a chemical would have no observable reproductive effect even if a person were exposed to 1000 times that level. We performed a comprehensive review of the literature, including the studies reviewed by DART-IC, and derived an oral MADL. Of all the studies we identified, Delclos et al. (2014) is of sufficient quality, has the lowest no observed effect level (NOEL), and results in the most conservative MADL of 157 µg/d. This is generally supported by other studies, including those that were considered by DART-IC. Also, the oral MADL provides a similar margin of safety as OEHHA's dermal MADL and other regulatory guidelines. Taken together, the scientific data support an oral MADL of 157 µg/d.

Subfertility caused by altered follicular development and oocyte growth in female mice lacking PKB alpha/Akt1.

Mammalian females are endowed with a finite number of primordial follicles at birth. Immediately following formation of the primordial follicle pool, cohorts of follicles are either culled from the ovary or are recruited to grow until the primordial follicle population is depleted. The majority of ovarian follicles, including the oocytes, undergo atresia through apoptotic cell death. As PKB alpha/Akt1 is known to regulate apoptosis, we asked whether Akt1 functioned in the regulation of folliculogenesis in the ovary. Akt1(-/-) females display reduced fertility and abnormal estrous cyclicity. At Postnatal Day (PND) 25, Akt1(-/-) ovaries possessed a reduced number of growing antral follicles, significantly larger primary and secondary oocytes, and an increase in the number of degenerate oocytes. By PND90, there was a significant decrease in the number of primordial follicles in Akt1(-/-) ovaries relative to Akt1(+/+). In vivo granulosa cell proliferation was reduced, as were expression levels of Kitl and Bcl2l1, two factors associated with granulosa cell proliferation/survival. No compensation was observed by Akt2 or Akt3 at the mRNA/protein level. Significantly higher serum LH and trends for lower FSH and higher inhibin A and lower inhibin B relative to Akt1(+/+) females were observed in Akt1(-/-) females. Exposure to exogenous gonadotropins resulted in an increase in the number of secondary follicles in Akt1(-/-) ovaries, but few mature follicles. Collectively, our results suggest that PKB alpha/Akt1 plays an instrumental role in the regulation of the growth and maturation of the ovary, and that the loss of PKB alpha/Akt1 results in premature ovarian failure.

Review of animal studies on the cardiovascular effects of caffeine.

To address the safety of caffeine levels in energy drinks, we previously conducted a detailed evaluation of epidemiology studies in humans consuming coffee/caffeine, in which we assessed multiple health effects (unpublished). To further evaluate the effects of caffeine on the cardiovascular system, we turned to animal studies, which often use pure caffeine (not coffee), frequently at higher doses than those typical of human exposure. We identified key scientific studies and reviews in which effects of coffee or caffeine were evaluated in animals by conducting a comprehensive PubMed literature search and analyzing the results. We found that the human equivalent dose (HED) for the no observed adverse effect level (NOAEL) for cardiovascular effects was 260 mg caffeine (2-3 cups of coffee) for a single dose of caffeine for a 70-kg adult, while the lowest observed adverse effect level (LOAEL) was 770 mg (7-8 cups of coffee) for a 70-kg adult. Overall, the doses associated with possible adverse cardiovascular effects were more than either the amount of caffeine consumed over a 24-hour period in two regular energy shots (400 mg/day) or the amount in two extra strength energy shots (460 mg/day).

Vascular smooth muscle polyploidization--from mitotic checkpoints to hypertension.

Aging and hypertension are accompanied by an increase in mass and rigidity of arterial walls. At capacitance arteries, the enlargement and stiffness of the medial smooth muscle layer promote systolic hypertension and contribute to left ventricular hypertrophy and cardiovascular morbidity. Morphological studies have demonstrated that vascular smooth muscle cell (VSMC) hypertrophy, with minimal hyperplasia, causes the enlargement of vascular smooth muscle at capacitance arteries, and that VSMC hypertrophy is strongly associated with VSMC polyploidization. Recent studies demonstrate that hypertrophic signals, such as those elicited by Angiotensin II, abrogate the mechanisms of control of M phase in VSMC and induce cell cycle re-entry and polyploidization. These polyploid VSMC have a lower replicative rate, but a higher mass, protein content and matrix production than their diploid counterparts. Both, the protein kinase Aktl and the cyclin kinase-associated protein CKsl, have been implicated in the mechanism of VSMC polyploidization during hypertension. Here, we review the function of these proteins at the mitotic spindle cell cycle checkpoint and their role in the process of VSMC polyploidization.

Loss of mitotic spindle checkpoint activity predisposes to chromosomal instability at early stages of fibrosarcoma development.

Mice carrying the bovine papillomavirus type I genome develop dermal fibrosarcomas in a multiple step process characterized by distinctive proliferative stages. Chromosomal aberrations are identified early in this tumorigenic pathway, however, the mechanism that originates them is unknown. Using a functional assay, we investigated the status of the mitotic spindle cell cycle checkpoint (MSCCC) that regulates the metaphase to anaphase transition, in cells representing different stages of fibrosarcoma progression. Loss of MSCCC activity was apparent in mild fibromatosis and completely abolished in aggressive fibromatosis and fibrosarcoma lesions. This altered MSCCC status was confirmed biochemically by deregulated expression of Cks1 protein and unscheduled cyclin B metabolism. Immunoprecipitation and sequencing analyses indicated that mutation of p53 was not required for the abrogation of the MSCCC. These results demonstrate that loss of mitotic spindle checkpoint activity predisposes to chromosomal instability at early stages of fibrosarcoma development. To our knowledge, these studies constitute the first report of a transition in MSCCC activity in a tumorigenesis model.

Expression and localization of total Akt1 and phosphorylated Akt1 in the rat seminiferous epithelium.

Akt1 is a survival factor that is believed to play a role in the transcriptional modulation of a subset of genes associated with cell growth, proliferation, and apoptosis. We have explored in detail the expression of total Akt1 and phosphorylated Akt1 in the developing and adult rat testis. Throughout testis postnatal development, the expression of total Akt1 protein exhibited a mainly cytoplasmic localization within both the germ cells and the supporting Sertoli cells. In contrast, phosphorylated Akt1 staining demonstrated a mainly nuclear localization within germ cells. In the developmental sequence of germ cells, phosphorylated Akt1 stained the nuclei of spermatogonia, spermatocytes, and round spermatids. During spermiogenesis, phosphorylated Akt1 staining decreased in the nucleus and became localized to a bright spot at the base of the nucleus in elongate spermatids. Of interest, total Akt1 was found to localize to the perinuclear region of germ cells and the supranuclear region of Sertoli cells, depending on fixation. Further analysis demonstrated this staining to be associated with the Golgi complex in both germ and Sertoli cells.

Reproductive effects in F1 adult females exposed in utero to moderate to high doses of mono-2-ethylhexylphthalate (MEHP).

Phthalates are widely used as plasticizers in everyday products. Yet, studies on the effects of phthalates on female reproductive health are limited. In this study, pregnant C57/Bl6 mice were exposed via oral gavage to corn oil, 100, 500, or 1000mg/kg MEHP from gestational days 17-19. Reproductive lifespan was decreased by one month in the highest F1 exposure group (9.8±0.4 versus 11.1±0.6 months in control F1 females). F1 females exhibited delayed estrous onset at the two higher exposures and prolonged estrus was observed in all MEHP-exposed females. Serum FSH and estradiol were significantly elevated at the highest exposure and altered mRNA expression was found for the steroidogenic genes LHCGR, aromatase, and StAR. At one year of age, mammary gland hyperplasia was observed in high dose MEHP-exposed females. In summary, late gestational exposure to MEHP leads to multiple latent reproductive effects throughout murine life resulting in premature ovarian senescence and mammary hyperplasia.

Akt1 protects against germ cell apoptosis in the postnatal mouse testis following lactational exposure to 6-N-propylthiouracil.

Exposure to 6-propyl-2-thio-uracil (PTU), a neonatal goitrogen, leads to increased testis size and sperm production in rodents. Akt1, a gene involved in cell survival and proliferation is also phosphorylated by thyroxine (T(4)). Therefore, we examined the requirement for Akt1 in germ cell survival following PTU-induced hypothyroidism. Experiments were performed using Akt1+/+, Akt1+/-, and Akt1-/- mice. PTU was administered (0.01% w/v) via the drinking water of dams from birth to PND21. At PND15, T(4) serum levels were similar in all control groups, and significantly lower in all exposed groups with a dramatic decrease in Akt1-/- mice. PTU-exposed Akt1-/- testes displayed smaller tubules, increased apoptosis, delayed lumen formation, and increased inhibin B and AMH mRNA. Relative adult testis weights were similar in all exposure groups; however, no increase in daily sperm production was observed in PTU-exposed Akt1-/- mice. In conclusion, Akt1 contributes to the effects of thyroid hormone on postnatal testis development.

Preliminary efficacy of the anti-insulin-like growth factor type 1 receptor antibody figitumumab in patients with refractory Ewing sarcoma.

PURPOSE: Patients with Ewing sarcoma (ES) with metastases and those who relapse fare poorly and receive therapies that carry significant toxicity. This phase 1/2 study was conducted to evaluate the efficacy of figitumumab in advanced ES. PATIENTS AND METHODS: Patients with sarcoma 10 to 18 years old were enrolled in two dose escalation cohorts (20 and 30 mg/Kg intravenously every 4 weeks) in the phase 1 portion of the study. Patients with ES 10 years old or older were enrolled in the phase 2 portion of the study. The primary phase 2 objective was objective response rate (ORR). RESULTS: Thirty-one patients with ES (n = 16), osteosarcoma (n = 11), or other sarcomas (n = 4) were enrolled in the phase 1 portion of the study. Dose escalation proceeded to 30 mg/kg every 4 weeks with no dose-limiting toxicity identified. In the phase 2 portion of the study, 107 patients with ES received figitumumab at 30 mg/kg every 4 weeks for a median of 2 cycles (range, 1 to 16). Sixty three percent of phase 2 patients had received at least three prior treatment regimens. Of 106 evaluable patients, 15 had a partial response (ORR, 14.2%) and 25 had stable disease. Median overall survival was 8.9 months. Importantly, patients with a pretreatment circulating free insulin-like growth factor (IGF) -1 lower than 0.65 ng/mL (n = 14) had a median OS of 3.6 months, whereas those with a baseline free IGF-1 ≥ 0.65 ng/mL (n = 84) had a median OS of 10.4 months (P < .001). CONCLUSION: Figitumumab had modest activity as single agent in advanced ES. A strong association between pretreatment serum IGF-1 and survival benefit was identified.

Development of inhibitors of the IGF-IR/PI3K/Akt/mTOR pathway.

Progress has been made towards the development of agents targeting tyrosine kinase receptors and other molecules involved in signalling pathways important for cell proliferation, motility, and apoptosis. Inhibitor molecules designed to be highly specific with the aim of decreasing toxicity have proven to be generally well tolerated. However, the efficacy of targeted agents may be impacted by cross-talk between pathways and downregulation of negative feed-back loops. That is the case of the IGF-IR/PI3K/Akt/mTOR pathway. This issue raises the question of how these targeted agents could be combined to prevent or delay resistance without significantly increasing toxicity. Several mTOR inhibitors have been approved for cancer therapy, and late-stage clinical trials of IGF-IR inhibitors are underway. The outcome of ongoing clinical studies of IGF-IR, PI3K, Akt and mTOR inhibitors as well as further testing of the combination of these agents will be key for the development of therapeutic options in a wide range of oncology indications.

Molecular analysis of non-small cell lung cancer identifies subsets with different sensitivity to insulin-like growth factor I receptor inhibition.

PURPOSE: This study aimed to identify molecular determinants of sensitivity of non-small cell lung cancer (NSCLC) to anti-insulin-like growth factor receptor (IGF-IR) therapy. EXPERIMENTAL DESIGN: A total of 216 tumor samples were investigated, of which 165 consisted of retrospective analyses of banked tissue and an additional 51 were from patients enrolled in a phase II study of figitumumab, a monoclonal antibody against IGF-IR, in stage IIIb/IV NSCLC. Biomarkers assessed included IGF-IR, epidermal growth factor receptor, IGF-II, IGF-IIR, insulin receptor substrate 1 (IRS-1), IRS-2, vimentin, and E-cadherin. Subcellular localization of IRS-1 and phosphorylation levels of mitogen-activated protein kinase and Akt1 were also analyzed. RESULTS: IGF-IR was differentially expressed across histologic subtypes (P = 0.04), with highest levels observed in squamous cell tumors. Elevated IGF-IR expression was also observed in a small number of squamous cell tumors responding to chemotherapy combined with figitumumab (P = 0.008). Because no other biomarker/response interaction was observed using classical histologic subtyping, a molecular approach was undertaken to segment NSCLC into mechanism-based subpopulations. Principal component analysis and unsupervised Bayesian clustering identified three NSCLC subsets that resembled the steps of the epithelial to mesenchymal transition: E-cadherin high/IRS-1 low (epithelial-like), E-cadherin intermediate/IRS-1 high (transitional), and E-cadherin low/IRS-1 low (mesenchymal-like). Several markers of the IGF-IR pathway were overexpressed in the transitional subset. Furthermore, a higher response rate to the combination of chemotherapy and figitumumab was observed in transitional tumors (71%) compared with those in the mesenchymal-like subset (32%; P = 0.03). Only one epithelial-like tumor was identified in the phase II study, suggesting that advanced NSCLC has undergone significant dedifferentiation at diagnosis. CONCLUSION: NSCLC comprises molecular subsets with differential sensitivity to IGF-IR inhibition.

Safety, pharmacokinetics, and pharmacodynamics of the insulin-like growth factor type 1 receptor inhibitor figitumumab (CP-751,871) in combination with paclitaxel and carboplatin.

INTRODUCTION: This phase 1 study was conducted to determine the recommended phase 2 dose of the selective insulin-like growth factor type 1 receptor (IGF-IR) inhibitor figitumumab (F, CP-751,871) given in combination with paclitaxel and carboplatin in patients with advanced solid tumors. METHODS: Patients received paclitaxel 200 mg/m2, carboplatin (area under the curve of 6), and F (0.05-20 mg/kg) q3 weeks for up to six cycles. Patients with objective response or stable disease were eligible to receive additional cycles of single agent F until disease progression. Safety, efficacy, pharmacokinetic, and pharmacodynamic endpoints were investigated. RESULTS: Forty-two patients, including 35 with stages IIIB and IV non-small cell lung cancer (NSCLC), were enrolled in eight dose escalation cohorts. A maximum tolerated dose was not identified. Severe adverse events possibly related to F included fatigue, diarrhea, hyperglycemia, gamma glutamyl transpeptidase elevation, and thrombocytopenia (one case each). F plasma exposure parameters increased with dose. Fifteen objective responses (RECIST) were reported, including two complete responses in NSCLC and ovarian carcinoma. Notably, levels of bioactive IGF-1 seemed to influence response to treatment with objective responses in patients with a high baseline-free IGF-1 to IGF binding protein-3 ratio seen only in the 10 and 20 mg/kg dosing cohorts. CONCLUSIONS: F was well tolerated in combination with paclitaxel and carboplatin. Based on its favorable safety, pharmacokinetic, and pharmacodynamic properties, the maximal feasible dose of 20 mg/kg has been selected for further investigation.