Bloom syndrome DNA helicase mitigates mismatch repair-dependent apoptosis.

Generation of O6-methylguanine (O6-meG) by DNA-alkylating agents such as N-methyl N-nitrosourea (MNU) activates the multiprotein mismatch repair (MMR) complex and the checkpoint response involving ATR/CHK1 and ATM/CHK2 kinases, which may in turn trigger cell cycle arrest and apoptosis. The Bloom syndrome DNA helicase BLM interacts with the MMR complex, suggesting functional relevance to repair and checkpoint responses. We observed a strong interaction of BLM with MMR proteins in HeLa cells upon treatment with MNU as evidenced by co-immunoprecipitation as well as colocalization in the nucleus as revealed by dual immunofluorescence staining. Knockout of BLM sensitized HeLa MR cells to MNU-induced cell cycle disruption and enhanced expression of the apoptosis markers cleaved caspase-9 and PARP1. MNU-treated BLM-deficient cells also exhibited a greater number of 53BP1 foci and greater phosphorylation levels of H2AX at S139 and RPA32 at S8, indicating the accumulation of DNA double-strand breaks. These findings suggest that BLM prevents double-strand DNA breaks during the MMR-dependent DNA damage response and mitigates O6-meG-induced apoptosis.

Chemopreventive effects of atractylenolide-III on mammary tumorigenesis via activation of the Nrf2/ARE pathway through autophagic degradation of Keap1.

The incidence of breast cancer is increasing annually, making it a major health threat for women. Chemoprevention using natural, dietary, or synthetic products has emerged as a promising approach to address this growing burden. Atractylenolide-III (AT-III), a sesquiterpenoid present in various medicinal herbs, has demonstrated potential therapeutic effects against several diseases, including tumors, nonalcoholic fatty liver disease, and cerebral ischemic injury. However, its impact on breast cancer chemoprevention remains unexplored. In this study, we used an N-methyl-N-nitrosourea (NMU)-induced rat breast cancer model and 17ß-estradiol (E2)-treated MCF-10A cells to evaluate the chemopreventive potential of AT-III on mammary tumorigenesis. AT-III inhibited mammary tumor progression, evidenced by reduced tumor volume and multiplicity, prolonged tumor latency, and the reversal of NMU-induced weight loss. Furthermore, AT-III suppressed NMU-induced inflammation and oxidative stress through the Nrf2/ARE pathway in breast cancer tissues. In vitro, AT-III effectively suppressed E2-induced anchorage-independent growth and cell migration in MCF-10A cells. Nrf2 knockdown attenuated the protective effects of AT-III, highlighting the pivotal role of Nrf2 in AT-III-mediated suppression of tumorigenesis. The mechanism involves the induction of Nrf2 expression by AT-III through the autophagic degradation of Kelch-like ECH-associated protein 1 (Keap1). Overall, the results of this study indicate that AT-III is a promising candidate for breast cancer chemoprevention and provide valuable insights into its molecular interactions and signaling pathways.

FANCD2 counteracts O6-methylguanine-induced mismatch repair-dependent apoptosis.

BACKGROUND: Sn1-type alkylating agents methylate the oxygen atom on guanine bases thereby producing O6-methylguanine. This modified base could pair with thymine and cytosine, resulting in the formation of O6-methylguanine/thymine mismatch during DNA replication, recognized by the mismatch repair (MMR) complex, which then initiates the DNA damage response and subsequent apoptotic processes. In our investigation of the molecular mechanisms underlying MMR-dependent apoptosis, we observed FANCD2 modification upon the activity of alkylating agent N-methyl-N-nitrosourea (MNU). This observation led us to hypothesize a relevant role for FANCD2 in the apoptosis induction process. METHODS AND RESULTS: We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci. CONCLUSIONS: Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.

Rev1 overexpression accelerates N-methyl-N-nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis.

Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.

YTHDF1 mediates N-methyl-N-nitrosourea-induced gastric carcinogenesis by controlling HSPH1 translation.

YT521-B homology (YTH) domain family (YTHDF) proteins serve as readers that directly recognise m6A modifications. In this study, we aim to probe the role of YTHDF1 in environmental carcinogen-induced malignant transformation of gastric cells and gastric cancer (GC) carcinogenesis. We established a long-term low-dose MNU-induced malignant transformation model in gastric epithelial cells. In vivo and in vitro experiments were conducted to validate the malignant phenotype and characterise the roles of YTHDF1 and its downstream genes in malignant transformation cells. Additionally, we explored downstream m6A modification targets of YTHDF1 using RNA-sequencing, RNA immunoprecipitation, and proteomics analyses, and conducted validation experiments in cell experiments and clinical samples. Long-term low-dose exposure of MNU converted normal Gges-1 cells into malignant cells. YTHDF1 mRNA and protein expression are increased in MNU-induced malignant cells (p<0.001). Meanwhile, YTHDF1 knockdown inhibits the malignant potential of MNU-treated cells (p<0.01). YTHDF1 knockdown specifically suppresses HSPH1 protein, but not RNA levels. RIP-qPCR validates HSPH1 is the target of YTHDF1 (p<0.01). HSPH1 knockdown impairs the malignant potential of MNU-induced transformed cells. The increased expression of the key regulatory factor YTHDF1 in MNU-induced gastric carcinogenesis affects malignant transformation and tumorigenesis by regulating the translation of downstream HSPH1. These findings provide new potential targets for preventing and treating environmental chemical-induced gastric carcinogenesis.

Effects of different alkylating agents on photoreceptor degeneration and proliferative response of Müller glia.

Animal models for retinal degeneration are essential for elucidating its pathogenesis and developing new therapeutic strategies in humans. N-methyl-N-nitrosourea (MNU) has been extensively used to construct a photoreceptor-specific degeneration model, which has served to unveil the molecular process of photoreceptor degeneration as well as the mechanisms regulating the protective responses of remaining cells. Methyl methanesulphonate (MMS), also known to cause photoreceptor degeneration, is considered a good alternative to MNU due to its higher usability; however, detailed pathophysiological processes after MMS treatment remain uncharacterized. Here, we analyzed the time course of photoreceptor degeneration, Müller glial proliferation, and expression of secretory factors after MNU and MMS treatments in rats. While the timing of rod degeneration was similar between the treatments, we unexpectedly found that cones survived slightly longer after MMS treatment. Müller glia reentered the cell cycle at a similar timing after the two treatments; however, the G1/S transition occurred earlier after MMS treatment. Moreover, growth factors such as FGF2 and LIF were more highly upregulated in the MMS model. These data suggest that comparative analyses of the two injury models may be beneficial for understanding the complex regulatory mechanisms underlying the proliferative response of Müller glia.

Hyperoside alleviates photoreceptor degeneration by preventing cell senescence through AMPK-ULK1 signaling.

Vision loss and blindness are frequently caused by photoreceptor degeneration, for example in age-related macular degeneration and retinitis pigmentosa. However, there is no effective medicine to treat these photoreceptor degeneration-related diseases. Cell senescence is a common phenotype in many diseases; however, few studies have reported whether it occurs in photoreceptor degeneration diseases. Herein, we identified that cell senescence is associated with photoreceptor degeneration induced by N-methyl-N-nitrosourea (MNU, a commonly used photoreceptor degeneration model), presented as increased senescence-associated ß-galactosidase activity, DNA damage, oxidative stress and inflammation-related cytokine Interleukin 6 (IL6), and upregulation of cyclin p21 or p16. These results suggested that visual function might be protected using anti-aging treatment. Furthermore, Hyperoside is reported to help prevent aging in various organs. In this study, we showed that Hyperoside, delivered intravitreally, alleviated photoreceptor cell senescence and ameliorated the functional and morphological degeneration of the retina in vivo and in vitro. Importantly, Hyperoside attenuated the MNU-induced injury and aging of photoreceptors via AMPK-ULK1 signaling inhibition. Taken together, our results demonstrated that Hyperoside can prevent MNU-induced photoreceptor degeneration by inhibiting cell senescence via the AMPK-ULK1 pathway.

Lutein combined with EGCG improved retinitis pigmentosa against N-methyl-N nitrosourea-induced.

In order to investigate the synergistic improving effect of lutein (LUT) and epigallocatechin-3-gallate (EGCG) treatment on retinitis pigmentosa (RP), an N-methyl-N-nitrosourea (MNU)-induced mouse model was conducted in the present study. Compared to the LUT alone treatment group, in the LUT combined with EGCG (LUT-EGCG) treatment group, the accumulation content of LUT was significantly increased by 50.24% in the liver. The morphological results indicated that LUT-EGCG treatment significantly improved the retina structure with the thickness of the outer nuclear layer restored to 185.28 ± 0.29 µm, showing no significant difference compared to the control group. The LUT-EGCG treatment also increased the production of short-chain fatty acids, such as acetic and propionic acids. Compared with the LUT alone treatment, the LUT-EGCG treatment significantly increased the relative abundance of Lachnospiraceae and Helicobacteraceae. RT-qPCR results indicated that LUT-EGCG treatment significantly increased the antiapoptotic gene Bcl-2 expression. In addition, the expression of IL-6 was significantly down-regulated in the LUT-EGCG group, while there was no significance in NF-κß, TNF-α, IL-1ß, and IL-18 compared with the LUT group. Correlation analysis supported the conclusion that LUT combined with EGCG may improve RP by modulating antiapoptotic gene expression and regulating the abundance of gut microbiota. However, the underlying mechanism still needs further research.

Short-term carcinogenicity study of N-methyl-N-nitrosourea in FVB-Trp53 heterozygous mice.

Carcinogenicity tests predict the tumorigenic potential of various substances in the human body by studying tumor induction in experimental animals. There is a need for studies that explore the use of FVB/N-Trp53em2Hwl/Korl (FVB-Trp53+/-) mice, created by TALEN-mediated gene targeting in Korea, in carcinogenicity tests. This study was performed to determine whether FVB-Trp53+/- mice are a suitable model for short-term carcinogenicity studies. To compare the carcinogenicity at different concentrations, 25, 50, and 75 mg/kg of N-methyl-N-nitrosourea (MNU), a known carcinogen, were administered intraperitoneally to FVB-Trp53+/- and wild-type male mice. After 26 weeks, the survival rate was significantly reduced in FVB-Trp53+/- mice compared to the wild-type mice in the 50 and 75 mg/kg groups. The incidence of thymic malignant lymphoma (TML) in the 50 and 75 mg/kg groups was 54.2 and 59.1% in FVB-Trp53+/- male mice, respectively. TML metastasized to the lungs, spleen, lymph nodes, liver, kidney, and heart in FVB-Trp53+/- male mice. Furthermore, the incidence of primary lung tumors, such as adenomas and adenocarcinomas, was 65.4, 62.5, and 45.4% in the FVB-Trp53+/- mice of the 25, 50, and 75 mg/kg groups, respectively. The main tumor types in FVB-Trp53+/- mice were TML and primary lung tumors, regardless of the dose of MNU administered. These results suggest that systemic tumors may result from malfunctions in the p53 gene and pathway, which is an important factor in the pathogenesis of human cancers. Therefore, FVB-Trp53 heterozygous mice are suitable for short-term carcinogenicity tests using positive carcinogens, and that the best result using MNU, a positive carcinogen, might have a single dose of 50 mg/kg.

Appraising Animal Models of Prostate Cancer for Translational Research: Future Directions.

The growing incidence of prostate cancer has prompted a great investment in basic biology and translational studies to develop new therapies. Multiple animal models have been established to study etiological factors, cancer-preventive strategies and the molecular determinants of aggressiveness and metastases. The rat model of prostate cancer induced by chemical carcinogen N-methyl-N-nitrosourea (MNU) and testosterone exposure has become an important tool to study prostatic carcinogenesis and chemopreventive approaches. Over prolonged treatment, this model develops prostatic lesions that closely mimic those observed in human patients. By modifying the experimental conditions, different research groups have been able to induce a vast spectrum of lesions, ranging from early prostatic intraepithelial neoplasia to metastatic cancer. These carefully tuned experimental settings allowed researchers to test lifestyle interventions, and different pharmacological and chemopreventive strategies. However, this model's great flexibility requires careful planning to ensure that the experimental conditions are adequate to obtain the spectrum of lesions intended. The present review addresses such issues, highlighting the value of the rat prostate cancer model and the multiple challenges and opportunities it offers to researchers worldwide.

Effects of perinatal exposure to bisphenol A on induction of prostate cancer in Sprague Dawley rats by MNU and testosterone.

Perinatal and neonatal exposure to bisphenol A (BPA) has been linked to enhancement of prostate carcinogenesis in rats induced by combined treatment with estradiol and testosterone, but human data are lacking. This study aimed to determine the effects of perinatal BPA exposure on induction of prostate cancer in rats by sequential treatment with N-methyl-N-nitrosamine (MNU) and continuous low dose administration of testosterone. Pregnant Sprague Dawley rats were exposed to BPA administered by subcutaneous Alzet minipumps at doses of 2.5 or 25 µg/kg body weight/day from gestational day 9 until postnatal day 28 when pups were weaned providing exposure of offspring in utero and via the mother's milk. At 10-12 weeks of age, one male offspring per litter was treated with an intraperitoneal injection of MNU after hormonal stimulation of prostatic cell proliferation followed two weeks later by subcutaneous insertion of Silastic implants containing testosterone until the termination of the study 57-58 weeks after MNU injection. The perinatal BPA exposure did not significantly affect the incidence of prostate carcinomas which was slightly lower in exposed rats (33-23 %) than in control animals (40 %). Carcinomas in all accessory sex glands combined were also insignificantly less frequent in exposed (46-48 %) than in control rats (60 %). The incidence of malignant tumors at any site in the body was significantly lower in exposed rats (81-65 %) than in controls (93 %). In conclusion, perinatal BPA exposure did not significantly modify prostate cancer induction by MNU plus testosterone in rats, unlike the enhancement of prostate carcinogenesis induced by treatments involving estradiol administration. Which of the two models of prostate carcinogenesis is more relevant for the human situation is unclear at present.

Evaluation of the cytotoxic, anticancer, and genotoxic activities of Acacia nilotica flowers and their effects on N-methyl-N-nitrosourea-induced genotoxicity in mice.

PURPOSE: In this study, two main research objectives were examined: (1) the cytotoxic and anticancer activities of the aqueous methanol extract from Acacia nilotica flowers on three human cancer cells, namely lung A549, breast MCF-7, and leukemia THP-1 cells, and (2) the genotoxic effects of A. nilotica extract and its influence on DNA damage induced by N-methyl-N-nitrosourea (MNU) in mice. METHODS: Mice were orally treated with A. nilotica extract (200, 500, and 800 mg/kg for 4 days) with or without MNU (80 mg/kg intraperitoneally for 24 h). RESULTS: In vitro experiments showed that A549 cells were the most sensitive to A. nilotica extract among the tested cell lines. A. nilotica extract inhibited A549 cell proliferation by blocking the cell cycle at the G2/M phase and accumulating apoptotic cells in the sub-G0/G1 phase in A549 cells. In vivo experiments showed that MNU induced positive and negative genotoxicity in bone marrow cells and spermatocytes, respectively. Negative genotoxicity was observed in A. nilotica extract-treated groups only. However, A. nilotica extract (800 mg/kg) remarkably increased comet tail formation in bone marrow cells. Unexpectedly, the absence of antigenotoxicity was observed in three cotreated groups with A. nilotica extract and MNU compared with the MNU-treated group. Astonishingly, cotreatment with MNU and A. nilotica extract at a dose above 200 mg/kg remarkably increased micronucleus and comet tail formation in bone marrow cells compared with the MNU-treated group. CONCLUSIONS: A. nilotica extract possessed anticancer activity with relative genotoxic effects at high doses.

Castration immunoregulates toll-like receptor-4 in male bladder cancer.

PURPOSE: Among diverse Pattern Recognition Receptors (PRRs), Toll-like receptor-4 (TLR-4) is a key urothelial trigger for innate immune response impacting urothelial bladder carcinoma (BC). Androgen activation promotes immunotolerance, playing an immunoregulatory role by unknown mechanisms. We explored the castration impact on urothelial TLR-4 modulation in carcinogenesis and immunotherapeutic scenario. METHODS: Intact (SHAM) versus castrated male Fisher-344 rats were evaluated in 2 scenarios: (A) Carcinogenesis: After randomization to SHAM (n = 5) and Castration (n = 5), carcinogenesis was induced by four intravesical doses of 1.5 mg/kg n-methyl-n-nitrosourea (MNU) every 15 days. (B) Treatment: After ultrasonographic confirmed MNU-induced papillary BC on week 8, rats were randomized to SHAM (n = 5) and Castration (n = 5) and offered 6 weekly intravesical treatment of 106 CFU of bacillus Calmette Guerin (BCG) in 0.2 ml saline. After 15 weeks the urinary bladders underwent histopathology. Urothelial cell proliferation was measured by Ki-67 immunohistochemistry (IHC), and TLR-4 expression was quantified by IHC and WB. RESULTS: Castration induced higher TLR-4 urothelial expression (p = 0.007) and anticarcinogenic effect with fewer urothelial tumors (60 vs. 80%) and lower urothelial cell proliferation compared to intact animals (p = 0.008). In the intravesical BCG treatment setting, castration has potentialized the BCG activation of TLR-4 (p = 0.007) with no residual in situ carcinoma compared to intact animals, suggesting the potential to amplify the BCG immune response. CONCLUSION: To our knowledge, this is the first description of TLR-4 urothelial expression hormonal modulation. The described castration-mediated immunomodulation will help to improve the knowledge of urothelial cancer gender diversities and PRRs modulations with treatment implications.

N-methyl-N-nitrosourea induces zebrafish anomalous angiogenesis through Wnt/ß-catenin pathway.

N-methyl-N-nitrosourea (MNU) is a prevalent environmental carcinogen, which leads to tumors in various organs in animal models, while the mechanisms involved were still not fully understood. It is well known that anomalous angiogenesis is a key step in tumorigenesis and progression. In this study, we found that MNU induced abnormal angiogenesis which was accompanied by upregulation of rspo1, p53 and vegfaa in zebrafish embryos. Moreover, it revealed that MNU-induced ectopic sprouting of blood vessels was significantly reduced in rspo1-knockdown but not p53-knockdown embryos, indicating that rspo1 was necessary for MNU-induced abnormal angiogenesis. Additionally, pharmaceutical activation or inhibition of Wnt/ß-catenin signaling pathway using (2'Z,3'E)- 6-bromoindirubin-3'-oxime or CCT036477 significantly increased or inhibited the pro-angiogenic effect of MNU on developing zebrafish embryos, which was confirmed by the effect of proliferation and migration in MNU-treated bEnd.3 cells. These data together indicated that rspo1/Wnt/ß-catenin/vegfaa axis is involved in the modulation of MNU-induced anomalous angiogenesis.

Copenhagen Rats Display Dominantly Inherited Yet Non-uniform Resistance to Spontaneous, Radiation-induced, and Chemically-induced Mammary Carcinogenesis.

BACKGROUND/AIM: Genetic and environmental factors interact to dictate the risk of cancer, and animal models are expected to provide avenues for identifying such interactions. The aim of the study was to clarify the genetic susceptibility of Copenhagen rats to spontaneous, radiation-induced, and chemically-induced mammary carcinogenesis. MATERIALS AND METHODS: Female Copenhagen and Sprague- Dawley rats and their F1 hybrids were subjected at age 7 weeks to γ-irradiation or intraperitoneal injection with 1-methyl-1-nitrosourea or were not treated, and palpable mammary tumours were diagnosed histologically. Data were pooled with previous data acquired for both nontreated and irradiated Sprague-Dawley rats. RESULTS: Radiation and 1-methyl-1-nitrosourea both significantly increased the incidence of mammary cancer in all strains. Copenhagen and F1 rats displayed a significantly lower incidence than Sprague-Dawley rats in all groups, with relatively higher incidence after irradiation. F1 rats exhibited significantly higher mammary cancer incidence than Copenhagen rats in the nontreated, but not the treated, groups. The interaction of the strain and exposure effects was suggested to be quasi-multiplicative. CONCLUSION: Copenhagen rats display non-uniform resistance to spontaneous, radiation-induced, and chemically-induced mammary carcinogenesis with dominant inheritance over Sprague-Dawley rats.

Chloroform extract of Calliandra portoricensis inhibits tumourigenic effect of N-methyl-N-nitrosourea and benzo(a)pyrene in breast experimental cancer.

Calliandra portoricensis (C. portoricensis) is used in herbal homes in Nigeria to manage breast diseases. We investigated the anti-tumourigenic effects of chloroform extract of C. portoricensis (CP) in breast experimental cancer induced by N-methyl-N-nitrosourea (NMU) and benzo-(a)-pyrene (BaP). Fifty-six female rats were assigned into seven equal groups: Group 1 served as control, group 2 received NMU and BaP (50 mg/kg, each), groups 3 and 4 received [NMU + BaP] and treated with CP at 50 and 100 mg/kg, respectively. Group 5 received CP (100 mg/kg), group 6 received [NMU + BaP] and vincristine (0.5 mg/kg), while group 7 received vincristine (0.5 mg/kg). The NMU and BaP (i.p) were dissolved in normal saline and corn oil, respectively. The CP (oral) and vincristine (i.p) were given thrice and twice per week, respectively for 10 weeks. The [NMU + BaP] intoxication significantly decreased body weight gain by 32% while organo-somatic weight of mammary gland increased by 37%. Also, [NMU + BaP] decreased the activities of mammary catalase, glutathione-s-transferase, glutathione peroxidase, superoxide dismutase and total sulphurhydryl by 34%, 31%, 35%, 35% and 33%, respectively. The [NMU + BaP] increased inflammatory and oxidative stress markers; nitrite, lipid peroxidation and myeloperoxidase by 62%, 57% and 361%, respectively. Strong expression of BCL-2, IL-6, COX 2, ß-catenin and iNOS in [NMU + BaP]-administered rats were observed. Histology revealed glands with malignant epithelial cells and high nucleocytoplasm in [NMU + BaP] rats. Treatment with CP attenuated inflammation, apoptosis and restored cyto-architecture of mammary gland. Overall, CP abates mammary tumourigenesis by targeting cellular pathways of inflammation and apoptosis.

Electrical response of retinal ganglion cells in an N-methyl-N-nitrosourea-induced retinal degeneration porcine model.

Retinal prosthesis is regarded as the treatment for vision restoration in the blind with retinal degeneration (RD) due to the loss of photoreceptors. A strategy for retinal prosthesis is to electrically activate surviving neurons. The retina's response to electrical stimulation in a larger RD model has not been studied yet. Therefore, in this study, we investigated electrically evoked retinal responses in a previously validated N-methyl-N-nitrosourea (MNU)-induced porcine RD model. Electrically evoked responses were evaluated based on the number of retinal ganglion cell (RGC) spikes via multichannel recordings. Stimulation pulses were applied to degenerative and wild-type retinas with pulse modulation. Compared to wild-type retinas, degenerative retinas showed higher threshold values of pulse amplitude and pulse duration. The rate of increase in the number of RGC spikes relative to stimulus intensity was lower in degenerative retinas than in normal retinas. In severely degenerated retinas, few RGCs showed electrically evoked spikes. Our results suggest that the degenerative porcine retina requires a higher charge than the normal porcine retina. In the early stage of RD, it is easier to induce RGC spikes through electrical stimulation using retinal prosthesis; however, when the degeneration is severe, there may be difficulty recovering patient vision.

Microemulsion for Prolonged Release of Fenretinide in the Mammary Tissue and Prevention of Breast Cancer Development.

The need of pharmacological strategies to preclude breast cancer development motivated us to develop a non-aqueous microemulsion (ME) capable of forming a depot after administration in the mammary tissue and uptake of interstitial fluids for prolonged release of the retinoid fenretinide. The selected ME was composed of phosphatidylcholine/tricaprylin/propylene glycol (45:5:50, w/w/w) and presented a droplet diameter of 175.3 ± 8.9 nm. Upon water uptake, the ME transformed successively into a lamellar phase, gel, and a lamellar phase-containing emulsion in vitro as the water content increased and released 30% of fenretinide in vitro after 9 days. Consistent with the slow release, the ME formed a depot in cell cultures and increased fenretinide IC50 values by 68.3- and 13.2-fold in MCF-7 and T-47D cells compared to a solution, respectively. At non-cytotoxic concentrations, the ME reduced T-47D cell migration by 75.9% and spheroid growth, resulting in ∼30% smaller structures. The depot formed in vivo prolonged a fluorochrome release for 30 days without producing any sings of local irritation. In a preclinical model of chemically induced carcinogenesis, ME administration every 3 weeks for 3 months significantly reduced (4.7-fold) the incidence of breast tumors and increased type II collagen expression, which might contribute to limit spreading. These promising results support the potential ME applicability as a preventive therapy of breast cancer.

Prenatal exposure to a mixture of different phthalates increases the risk of mammary carcinogenesis in F1 female offspring.

Phthalates metabolites have been detected in the urine of pregnant and breastfeeding women. Thus, this study evaluated the adverse effects of maternal exposure to a mixture of six phthalates (Pth mix) on the mammary gland development and carcinogenesis in F1 female offspring. Pregnant female Sprague-Dawley rats were exposed daily to vehicle or Pth mix (35.22% diethyl-phthalate, 21.03% di-(2-ethylhexyl)-phthalate, 14.91% dibutyl-phthalate, 15.10% diisononyl-phthalate, 8.61% diisobutyl-phthalate, and 5.13% benzylbutyl-phthalate) by gavage at 20 µg/kg, 200 µg/kg or 200 mg/kg during gestational day 10 (GD 10) to postnatal day 21 (PND 21). After weaning (PND 22), some female offspring were euthanized for mammary gland analyses while other females received a single dose of N-methyl-N-nitrosourea (MNU, 50 mg/kg) or vehicle and then tumor incidence and multiplicity were recorded until PND 180. Maternal Pth mix exposure increased the number of Ki-67 and progesterone receptor-positive epithelial cells in the mammary gland from Pth mix 200 at µg/kg and 200 mg/kg groups. In addition, tumor incidence and mean number were higher only in Pth mix at 200 mg/kg when compared to the vehicle-treated group, and percentage of tumor-free animals was lower in Pth mix at 200 µg/kg and 200 mg/kg groups. The findings indicate that perinatal Pth mixture exposure increased susceptibility to MNU-induced mammary carcinogenesis in adult F1 female offspring.

Molecular Hydrogen Attenuated N-methyl-N-Nitrosourea Induced Corneal Endothelial Injury by Upregulating Anti-Apoptotic Pathway.

Purpose: Previous work by our group has demonstrated the value of N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation in animal models. The aim of this study was to investigate the effect of molecular hydrogen (H2) on MNU-induced corneal endothelial cell (CEC) injury and the underlying mechanism. Methods: MNU-induced animal models of CEC injury were washed with hydrogen-rich saline (HRS) for 14 days. Immunofluorescence staining, immunohistochemical staining, and corneal endothelial assessment were applied to determine architectural and cellular changes on the corneal endothelium following HRS treatment. MNU-induced cell models of CEC injury were co-cultured with H2. The effect of H2 was examined using morphological and functional assays. Results: It was shown that MNU could inhibit the proliferation and specific physiological functions of CECs by increasing apoptosis and decreasing the expression of ZO-1 and Na+/K+-ATPase, whereas H2 improved the proliferation and physiological function of CECs by anti-apoptosis. Cell experiments further confirmed that H2 could reverse MNU damage to CECs by decreasing oxidative stress injury, interfering with the NF-κB/NLRP3 pathway and the FOXO3a/p53/p21 pathway. Conclusions: This study suggests that topical application of H2 could protect CECs against corneal damage factors through anti-apoptotic effect, reduce the incidence and severity of corneal endothelial decompensation, and maintain corneal transparency.