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.

Life-Cycle-Dependent Toxicities of Mono- and Bifunctional Alkylating Agents in the 3R-Compliant Model Organism C. elegans.

Caenorhabditis elegans (C. elegans) is gaining recognition and importance as an organismic model for toxicity testing in line with the 3Rs principle (replace, reduce, refine). In this study, we explored the use of C. elegans to examine the toxicities of alkylating sulphur mustard analogues, specifically the monofunctional agent 2-chloroethyl-ethyl sulphide (CEES) and the bifunctional, crosslinking agent mechlorethamine (HN2). We exposed wild-type worms at different life cycle stages (from larvae L1 to adulthood day 10) to CEES or HN2 and scored their viability 24 h later. The susceptibility of C. elegans to CEES and HN2 paralleled that of human cells, with HN2 exhibiting higher toxicity than CEES, reflected in LC50 values in the high µM to low mM range. Importantly, the effects were dependent on the worms' developmental stage as well as organismic age: the highest susceptibility was observed in L1, whereas the lowest was observed in L4 worms. In adult worms, susceptibility to alkylating agents increased with advanced age, especially to HN2. To examine reproductive effects, L4 worms were exposed to CEES and HN2, and both the offspring and the percentage of unhatched eggs were assessed. Moreover, germline apoptosis was assessed by using ced-1p::GFP (MD701) worms. In contrast to concentrations that elicited low toxicities to L4 worms, CEES and HN2 were highly toxic to germline cells, manifesting as increased germline apoptosis as well as reduced offspring number and percentage of eggs hatched. Again, HN2 exhibited stronger effects than CEES. Compound specificity was also evident in toxicities to dopaminergic neurons-HN2 exposure affected expression of dopamine transporter DAT-1 (strain BY200) at lower concentrations than CEES, suggesting a higher neurotoxic effect. Mechanistically, nicotinamide adenine dinucleotide (NAD+) has been linked to mustard agent toxicities. Therefore, the NAD+-dependent system was investigated in the response to CEES and HN2 treatment. Overall NAD+ levels in worm extracts were revealed to be largely resistant to mustard exposure except for high concentrations, which lowered the NAD+ levels in L4 worms 24 h post-treatment. Interestingly, however, mutant worms lacking components of NAD+-dependent pathways involved in genome maintenance, namely pme-2, parg-2, and sirt-2.1 showed a higher and compound-specific susceptibility, indicating an active role of NAD+ in genotoxic stress response. In conclusion, the present results demonstrate that C. elegans represents an attractive model to study the toxicology of alkylating agents, which supports its use in mechanistic as well as intervention studies with major strength in the possibility to analyze toxicities at different life cycle stages.

Screening for urinary markers predicting hematopoietic stem cell injury induced by busulfan using genetically diverse mice.

BACKGROUND: Busulfan (BU) is an alkylating agent used as a conditioning agent prior to hematopoietic stem cell (HSC) transplantation as it is known to be cytotoxic to host hematopoietic stem and progenitor cells. The susceptibility of HSCs to BU injury plays an important role in the myeloablative efficacy of BU. Different susceptibilities were demonstrated in genetically diverse (GD) mice in our preliminary research. METHODS: Three strains of GD mice with different susceptibilities to BU-induced HSC injury were used for screening biological markers of HSC injury susceptibility in urine. The urine proteins were analyzed using liquid chromatography coupled with tandem mass spectrometry to screen for differentially expressed proteins. Screening for possible biomarkers based on differences in protein expression abundance was validated using enzyme-linked immunoassay (ELISA). RESULTS: Functional analysis showed that the differential proteins were all involved in a series of biological pathways related to cellular senescence, apoptosis, and angiogenesis; whereas the differential proteins of the high-susceptible strain were enriched for the regulation of bone marrow microenvironment pathways, those of low-susceptible strain were enriched for the proapoptotic effect of GTPase pathways. Based on protein abundance differences, several urinary proteins that may be indicative of susceptibility were screened, and ELISA validation results showed that angiotensin-converting enzyme may be a potential biomarker predicting HSC susceptibility for BU conditioning. CONCLUSIONS: This study indicates that urinary protein levels can reflect differences in susceptibility to BU-induced HSC injury. Using GD mice to construct genetic difference models will provide preclinical data for screening BU-related biological markers.

Loss of p16 does not protect against premature ovarian insufficiency caused by alkylating agents.

BACKGROUND: Chemical agents such as alkylating agents (AAs) that are commonly used for the treatment of cancer cause great damage to the ovaries, thereby significantly increasing the risk of premature ovarian insufficiency (POI). However, the exact molecules underlying AA-induced POI remain largely obscure. Upregulation of the p16 gene may contribute to the progression of POI. As yet, no in vivo data from p16-deficient (KO) mice are available to demonstrate a critical role of p16 in POI. In the present study, we employed p16 KO mice to investigate whether loss of p16 could protect against POI caused by AAs. METHODS: WT mice and their p16 KO littermates received a single dose of BUL + CTX to establish an AA-induced POI mouse model. One month later, oestrous cycles were monitored. Three months later, some of the mice were sacrificed to collect sera for measurements of hormone levels and ovaries for measurements of follicle counts, the proliferation and apoptosis of granulosa cells, ovarian stromal fibrosis and vessels. The remaining mice were mated with fertile males for the fertility test. RESULTS: Our results showed that treatment with BUL + CTX significantly disrupted the oestrous cycles, increased the levels of FSH and LH while decreasing the levels of E2 and AMH, decreased the counts of primordial follicles and growing follicles while increasing the counts of atretic follicles, reduced the vascularized area in the ovarian stroma, and decreased fertility. All of these results were comparable between WT and p16 KO mice treated with BUL + CTX. In addition, ovarian fibrosis was not increased significantly in WT and p16 KO mice treated with BUL + CTX. Growing follicles with normal appearance had normally proliferating granulosa cells (without apparent apoptosis). CONCLUSION: We concluded that genetic ablation of the p16 gene did not attenuate ovarian damage or help preserve the fertility of mice challenged by AAs. This study demonstrated for the first time that p16 is dispensable for AA-induced POI. Our preliminary findings suggest that targeting p16 alone may not preserve the ovarian reserve and fertility of females treated with AAs.

Activation of the human TRPA1 channel by different alkylating sulfur and nitrogen mustards and structurally related chemotherapeutic drugs.

An important target in toxicology is the ion channel known as human transient receptor potential ankyrin 1 (hTRPA1). It is triggered by a variety of chemicals, including the alkylating chemical warfare agent sulfur mustard (SM). The activation potentials of structural analogs including O- and sesquimustard, nitrogen mustards (HN1, HN2, and HN3), and related chemotherapeutic drugs (bendamustine, cycylophosphamide, and ifosfamide) were examined in the current study. The aequorin assay was used to measure changes in intracellular calcium levels in human hTRPA1 overexpressing HEK293 cells. The XTT assay was used to determine cytotoxicity. The data presented here highlight that all investigated alkylating substances, with the exception of cyclophosphamide and ifosfamide, cause the activation of hTRPA1. Cytotoxicity and activation of hTRPA1 were found to be related. Compounds with high reactivity had higher cytotoxicity and vice versa. However, inhibiting hTRPA1 with the specific inhibitor AP18 could not reduce the cytotoxicity induced by alkylating agents. As a result, hTRPA1 does not play a significant role in the cytotoxicity of alkylating agents.

PARP1-MGMT complex underpins pathway crosstalk in O6-methylguanine repair.

DNA lesions induced by alkylating agents are repaired by two canonical mechanisms, base excision repair dependent on poly(ADP) ribose polymerase 1 (PARP1) and the other mediated by O6-methylguanine (O6meG)-DNA methyltransferase (MGMT) in a single-step catalysis of alkyl-group removal. O6meG is the most cytotoxic and mutagenic lesion among the methyl adducts induced by alkylating agents. Although it can accomplish the dealkylation reaction all by itself as a single protein without associating with other repair proteins, evidence is accumulating that MGMT can form complexes with repair proteins and is highly regulated by a variety of post-translational modifications, such as phosphorylation, ubiquitination, and others. Here, we show that PARP1 and MGMT proteins interact directly in a non-catalytic manner, that MGMT is subject to PARylation by PARP1 after DNA damage, and that the O6meG repair is enhanced upon MGMT PARylation. We provide the first evidence for the direct DNA-independent PARP1-MGMT interaction. Further, PARP1 and MGMT proteins also interact via PARylation of MGMT leading to formation of a novel DNA damage inducible PARP1-MGMT protein complex. This catalytic interaction activates O6meG repair underpinning the functional crosstalk between base excision and MGMT-mediated DNA repair mechanisms. Furthermore, clinically relevant 'chronic' temozolomide exposure induced PARylation of MGMT and increased binding of PARP1 and MGMT to chromatin in cells. Thus, we provide the first mechanistic description of physical interaction between PARP1 and MGMT and their functional cooperation through PARylation for activation of O6meG repair. Hence, the PARP1-MGMT protein complex could be targeted for the development of advanced and more effective cancer therapeutics, particularly for cancers sensitive to PARP1 and MGMT inhibition.

Royal jelly reduce DNA damage induced by alkylating agent in mice.

Royal jelly (RJ) is a creamy white-yellow liquid that is secreted by the mandibular and hypopharyngeal glands of bees to nourish the larvae. RJ has gained increasing interest in recent years owing to its antioxidant potential. However, little is known about adequate RJ dosing and its effects on genetic material. Thus, the aim of this study was to evaluate the in vivo effects of RJ on genotoxicity and mutagenicity induced by the alkylating agent methyl methanesulfonate (MMS). In this study, 3-month-old Swiss albino male mice (N = 66) were divided into 11 groups for experimentation. Experiments were performed by administering lyophilized RJ (150 mg/kg, 300 mg/kg, and 1000 mg/kg) or water via gavage as pre- and posttreatment processes with the alkylating agent MMS. After treatment, blood samples were collected from the mice via an incision at the end of the tail to conduct comet assays at times of 24 h and 48 h posttreatment. The mice were then euthanized to remove the bone marrow for a micronucleus test. Overall, regardless of dose, RJ did not exhibit genotoxic, mutagenic activity and the administration of high doses, mainly in the form of posttreatment, presented antigenotoxic and antimutagenic actions. Further, a dose-response correlation was observed in the RJ posttreatment groups. These results demonstrate that RJ administration was effective in reversing the damage caused by the alkylating agent MMS.

Dual regulating of mitochondrial fusion and Timp-3 by leflunomide and diallyl disulfide combination suppresses diethylnitrosamine-induced hepatocellular tumorigenesis in rats.

AIMS: Hepatocellular carcinoma (HCC) is considered one of the main causes of cancer-related death globally. Combination therapy targeting different pathways can improve the efficacy of HCC management. Mitofusin 2 (Mfn2), a mitochondrial fusion protein, and a tissue inhibitor of matrix metalloproteinase 3 (Timp-3) were found to be downregulated in various cancers, including HCC. Our study aimed to evaluate the possible antineoplastic effect of a novel combination in the treatment of HCC through targeting mitochondrial fusion and metastatic proteins. MAIN METHODS: HCC induction was performed using a single intraperitoneal dose of diethylnitrosamine (200 mg/kg), followed by adding phenobarbital sodium (0.05%) to the drinking water for successive 18 weeks. Then, leflunomide (LF, 10 mg/kg) was administered orally for 28 days. Diallyl disulfide (DADS, 50 mg/kg) was also given orally for 28 days, either alone or in combination with LF. KEY FINDINGS: Treatment with LF or DADS could alleviate the HCC- induced histological and biochemical variations, including liver enzyme activities (ALT, AST), alpha-fetoprotein, Bax, cyclin D1, Ki67, malondialdehyde, and reduced glutathione. They could shift the mitochondrial dynamics toward mitochondrial fusion through upregulating the expression of Mfn2 and also exhibited antimetastatic activity through upregulating the expression of Timp-3 and decreasing hepatic MMP9 content. SIGNIFICANCE: the treatment with a combination of LF and DADS displayed a more potent effect than the treatment with each drug alone. Our results suggest that the combined use of LF and a naturally occurring DADS can be used as a promising novel combination in managing HCC.

Toxic blister agents: Chemistry, mode of their action and effective treatment strategies.

Since their use during the First World War, Blister agents have posed a major threat to the individuals and have caused around two million casualties. Major incidents occurred not only due to their use as chemical warfare agents but also because of occupational hazards. Therefore, a clear understanding of these agents and their mode of action is essential to develop effective decontamination and therapeutic strategies. The blister agents have been categorised on the basis of their chemistry and the biological interactions that entail post contamination. These compounds have been known to majorly cause blisters/bullae along with alkylation of the contaminated DNA. However, due to the high toxicity and restricted use, very little research has been conducted and a lot remains to be clearly understood about these compounds. Various decontamination solutions and detection technologies have been developed, which have proven to be effective for their timely mitigation. But a major hurdle seems to be the lack of proper understanding of the toxicological mechanism of action of these compounds. Current review is about the detailed and updated information on physical, chemical and biological aspects of various blister agents. It also illustrates the mechanism of their action, toxicological effects, detection technologies and possible decontamination strategies.

Mitomycin induces alveolar epithelial cell senescence by down-regulating GSK3ß signaling.

Mitomycin treatment induces pulmonary toxicity, and alveolar epithelial cell senescence is crucial in the pathogenesis of the latter. However, the mechanism by which mitomycin induces alveolar epithelial cell senescence has yet to be elucidated. In this work, different doses (37.5-300 nM) of mitomycin induced the senescence of human alveolar type II-like epithelial cells and enhanced the phosphorylation of GSK3ß (S9). The GSK3ß (S9A) mutant reversed the senescence of mitomycin-treated alveolar epithelial cells. Pharmacological inhibition and gene deletion of Akt1, a kinase that regulates the phosphorylation of GSK3ß (S9), suppressed mitomycin-induced alveolar epithelial cell senescence. The knockdown of p53, a downstream effector of GSK3ß and an important regulator of cell senescence, repressed mitomycin-induced alveolar epithelial cell senescence. Treatment with baicalein weakened the phosphorylation of GSK3ß (S9) and alleviated the senescence of alveolar epithelial cells brought about by mitomycin treatment. GSK3ß (S9) phosphorylation appears to be the first signal involved in the mitomycin-induced senescence of alveolar epithelial cells and may present a potential target for attenuating mitomycin-induced pulmonary toxicity.

MicroRNA-26a systemic administration attenuates tumor formation in hepatocellular carcinoma mouse model.

MicroRNA (miRNA)-26a is one of the tumor suppressor genes that has been down regulated during the development of hepatocellular carcinoma (HCC). This work was conducted to evaluate the possible preventive effect of exogenous miRNA-26a administration on diethylnitrosamine (DEN)-mediated HCC. Balb/C mice were intraperitoneally injected with saline (Normal group), DEN (HCC group) or miRNA-26a (HCC+miRNA-26a group). On week 8, 12, 16 and 20, the concentrations of alpha-fetoprotein (AFP), des-gamma carboxyprothrombin (DCP), the levels of helper T cells-associated cytokines, and the vascular endothelial growth factor (VEGF), were measured. Flow cytometry determined the frequencies of regulatory T (Treg) cells. The concentrations of AFP, DCP and VEGF, as well as the frequency of Treg cells showed significantly lower values following miRNA-26a administration than in HCC group. miRNA-26a administration has reduced the levels of IL (interleukin)-2 and TNF (tumor necrosis factor)-α, in contrast, IL-10 level was markedly elevated in comparison to HCC model at all experimental time points. The restore of miRNA-26a function significantly (P<0.001) down regulated the expression levels of survivin & caspase-3 compared to HCC group. The obtained data introduce an evidence for the suppressive impact of miRNA-26a on liver tumor formation and its possible manipulation as a therapeutic design for HCC.

Persistent mTORC1 activation via Depdc5 deletion results in spontaneous hepatocellular carcinoma but does not exacerbate carcinogen- and high-fat diet-induced hepatic carcinogenesis in mice.

The mechanistic target of rapamycin complex 1 (mTORC1) acts as a central regulator of metabolic pathways that drive cellular growth. Abnormal activation of mTORC1 occurs at high frequency in human and mouse hepatocellular carcinoma (HCC). DEP domain-containing protein 5 (DEPDC5), a component of GATOR1 complex, is a repressor of amino acid-sensing branch of the mTORC1 pathway. In the current study, we found that persistent activation of hepatic mTORC1 signaling caused by Depdc5 ablation was sufficient to induce a pathological program of liver damage, inflammation and fibrosis that triggers spontaneous HCC development. Take advantage of the combinatory treatment with a single dose of diethylnitrosamine (DEN) and chronic feeding with high-fat diet (HFD), we demonstrated that hepatic depdc5 deletion did not aggravate DEN&HFD induced liver tumorigenesis, probably due to its protective effects on diet-induced liver steatosis. In addition, we further showed that chronic rapamycin treatment did not have any apparent tumor-suppressing effects on DEN&HFD treated control mice, whereas it dramatically reduced the tumor burden in mice with hepatic Depdc5 ablation. This study provides the novel in vivo evidence for Depdc5 deletion mediated mTORC1 hyperactivation in liver tumorigenesis caused by aging or DEN&HFD treatment. Moreover, our findings also propose that pharmacological inhibition of mTORC1 signaling maybe a promising strategy to treat HCC patients with mutations in DEPDC5 gene.

The cyto-protective effects of LH on ovarian reserve and female fertility during exposure to gonadotoxic alkylating agents in an adult mouse model.

STUDY QUESTION: Does LH protect mouse oocytes and female fertility from alkylating chemotherapy? SUMMARY ANSWER: LH treatment before and during chemotherapy prevents detrimental effects on follicles and reproductive lifespan. WHAT IS KNOWN ALREADY: Chemotherapies can damage the ovary, resulting in premature ovarian failure and reduced fertility in cancer survivors. LH was recently suggested to protect prepubertal mouse follicles from chemotoxic effects of cisplatin treatment. STUDY DESIGN, SIZE, DURATION: This experimental study investigated LH effects on primordial follicles exposed to chemotherapy. Seven-week-old CD-1 female mice were randomly allocated to four experimental groups: Control (n = 13), chemotherapy (ChT, n = 15), ChT+LH-1x (n = 15), and ChT+LH-5x (n = 8). To induce primary ovarian insufficiency (POI), animals in the ChT and ChT+LH groups were intraperitoneally injected with 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan, while control mice received vehicle. For LH treatment, the ChT+LH-1x and ChT+LH-5x animals received a 1 or 5 IU LH dose, respectively, before chemotherapy, then a second LH injection administered with chemotherapy 24 h later. Then, two animals/group were euthanized at 12 and 24 h to investigate the early ovarian response to LH, while remaining mice were housed for 30 days to evaluate short- and long-term reproductive outcomes. The effects of LH and chemotherapy on growing-stage follicles were analyzed in a parallel experiment. Seven-week-old NOD-SCID female mice were allocated to control (n = 5), ChT (n = 5), and ChT+LH-1x (n = 6) groups. Animals were treated as described above, but maintained for 7 days before reproductive assessment. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the first experiment, follicular damage (phosphorylated H2AX histone (γH2AX) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay), apoptotic biomarkers (western blot), and DNA repair pathways (western blot and RT-qPCR) were assessed in ovaries collected at 12 and 24 h to determine early ovarian responses to LH. Thirty days after treatments, remaining mice were stimulated (10 IU of pregnant mare serum gonadotropin (PMSG) and 10 IU of hCG) and mated to collect ovaries, oocytes, and embryos. Histological analysis was performed on ovarian samples to investigate follicular populations and stromal status, and meiotic spindle and chromosome alignment was measured in oocytes by confocal microscopy. Long-term effects were monitored by assessing pregnancy rate and litter size during six consecutive breeding attempts. In the second experiment, mice were stimulated and mated 7 days after treatments and ovaries, oocytes, and embryos were collected. Follicular numbers, follicular protection (DNA damage and apoptosis by H2AX staining and TUNEL assay, respectively), and ovarian stroma were assessed. Oocyte quality was determined by confocal analysis. MAIN RESULTS AND THE ROLE OF CHANCE: LH treatment was sufficient to preserve ovarian reserve and follicular development, avoid atresia, and restore ovulation and meiotic spindle configuration in mature oocytes exposed at the primordial stage. LH improved the cumulative pregnancy rate and litter size in six consecutive breeding rounds, confirming the potential of LH treatment to preserve fertility. This protective effect appeared to be mediated by an enhanced early DNA repair response, via homologous recombination, and generation of anti-apoptotic signals in the ovary a few hours after injury with chemotherapy. This response ameliorated the chemotherapy-induced increase in DNA-damaged oocytes and apoptotic granulosa cells. LH treatment also protected growing follicles from chemotherapy. LH reversed the chemotherapy-induced depletion of primordial and primary follicular subpopulations, reduced oocyte DNA damage and granulosa cell apoptosis, restored mature oocyte cohort size, and improved meiotic spindle properties. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was a preliminary study performed with mouse ovarian samples. Therefore, preclinical research with human samples is required for validation. WIDER IMPLICATIONS OF THE FINDINGS: The current study tested if LH could protect the adult mouse ovarian reserve and reproductive lifespan from alkylating chemotherapy. These findings highlight the therapeutic potential of LH as a complementary non-surgical strategy for preserving fertility in female cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Regional Valencian Ministry of Education (PROMETEO/2018/137), the Spanish Ministry of Science and Innovation (CP19/00141), and the Spanish Ministry of Education, Culture and Sports (FPU16/05264). The authors declare no conflict of interest.

PacBio sequencing detects genome-wide ultra-low-frequency substitution mutations resulting from exposure to chemical mutagens.

Genetic toxicology uses several assays to identity mutagens and protects the public. Most of these assays, however, rely on reporter genes, can only measure mutation indirectly based on phenotype, and often require specific cell lines or animal models-features that impede their integration with existing and emerging toxicological models, such as organoids. In this study, we show that PacBio Single-Molecule, Real-Time (PB SMRT) sequencing identified substitution mutations caused by chemical mutagens in Escherichia coli by generating nearly error-free consensus reads after repeatedly inspecting both strands of circular DNA molecules. Using DNA from E. coli exposed to ethyl methanosulfonate (EMS) or N-ethyl-N-nitrosourea (ENU), PB SMRT sequencing detected mutation frequencies (MFs) and spectra comparable to those obtained by clone-sequencing from the same exposures. The optimized background MF of PB SMRT sequencing was ≤ 1 × 10-7 mutations per base pair (mut/bp).

Biomarkers of geno- and cytotoxicity in the native broad-snouted caiman (Caiman latirostris): Chromosomal aberrations and mitotic index.

We evaluated the sensitivity of the chromosomal aberration (CA) and mitotic index (MI) assays on peripheral blood lymphocytes (PBLs) of Caiman latirostris, following ex vivo exposure to the alkylating agent, MMS. Two concentrations of MMS were tested in cultured peripheral blood. Relative to controls, MMS exposure reduced the number of metaphases observed, but both the numbers of cells with MN and the percentages of aberrant metaphases increased. The types of CA identified were chromosome and chromatid breaks, chromosomal rearrangements, monosomies, and nullisomies, with significantly higher values in the MMS-exposed groups. The incorporation of the MI and CA tests in C. latirostris can provide information on damage caused by xenobiotic exposures.

Differential exposure to N-ethyl N-nitrosourea during pregnancy is relevant to the induction of glioma and PNSTs in the brain.

Exposure to N-nitroso compounds (NOCs) during pregnancy has been associated with an increase in brain tumors in the progeny. This study investigated the brain tumorigenic effect of N-ethyl N-nitrosourea (ENU) after differential exposure of rats during pregnancy. Sprague Dawley rats were exposed to a single dose of ENU (80 mg/kg) in three different circumstances: 1) at first, second or third week of gestation; 2) at the 15th embryonic day (E15) in consecutive litters and 3) at E15 in three successive generations. Location and characterization of the offspring's brain tumors were performed by magnetic resonance imaging and histopathological studies. Finally, tumor incidence and latency and the animals' survival were recorded. ENU-exposure in the last two weeks of pregnancy induced intracranial tumors in over 70% of the offspring rats, these being mainly gliomas with some peripheral nerve sheath tumors (PNSTs). Tumors appeared in young adults; glioma-like small multifocal neoplasias converged on large glioblastomas in senescence and PNSTs in the sheath of the trigeminal nerve, extending to cover the brain convexity. ENU-exposure at E15 in subsequent pregnancies lead to an increase in glioma and PNST incidence. However, consecutive generational ENU-exposure (E15) decreased the animals' survival due to an early onset of both types of tumors. Moreover, PNST presented an inheritable component because progeny, which were not themselves exposed to ENU but whose progenitors were, developed PNSTs. Our results suggest that repeated exposure to ENU later in pregnancy and in successive generations favours the development of intracranial gliomas and PNSTs in the offspring.

Myelocytomatosis-Protein Arginine N-Methyltransferase 5 Axis Defines the Tumorigenesis and Immune Response in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: HCC is a leading cause of cancer-related deaths globally with poor outcome and limited therapeutic options. Although the myelocytomatosis (MYC) oncogene is frequently dysregulated in HCC, it is thought to be undruggable. Thus, the current study aimed to identify the critical downstream metabolic network of MYC and develop therapies for MYC-driven HCC. APPROACH AND RESULTS: Liver cancer was induced in mice with hepatocyte-specific disruption of Myc and control mice by administration of diethylnitrosamine. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses revealed that urinary dimethylarginine, especially symmetric dimethylarginine (SDMA), was increased in the HCC mouse model in an MYC-dependent manner. Analyses of human samples demonstrated a similar induction of SDMA in the urines from patients with HCC. Mechanistically, Prmt5, encoding protein arginine N-methyltransferase 5, which catalyzes SDMA formation from arginine, was highly induced in HCC and identified as a direct MYC target gene. Moreover, GSK3326595, a PRMT5 inhibitor, suppressed the growth of liver tumors in human MYC-overexpressing transgenic mice that spontaneously develop HCC. Inhibition of PRMT5 exhibited antiproliferative activity through up-regulation of the tumor suppressor gene Cdkn1b/p27, encoding cyclin-dependent kinase inhibitor 1B. In addition, GSK3326595 induced lymphocyte infiltration and major histocompatibility complex class II expression, which might contribute to the enhanced antitumor immune response. Combination of GSK3326595 with anti-programed cell death protein 1 (PD-1) immune checkpoint therapy (ICT) improved therapeutic efficacy in HCC. CONCLUSIONS: This study reveals that PRMT5 is an epigenetic executer of MYC, leading to repression of the transcriptional regulation of downstream genes that promote hepatocellular carcinogenesis, highlights a mechanism-based therapeutic strategy for MYC-driven HCC by PRMT5 inhibition through synergistically suppressed proliferation and enhanced antitumor immunity, and finally provides an opportunity to mitigate the resistance of "immune-cold" tumor to ICT.

Photobiomodulation Therapy Improves Spermatogenesis in Busulfan-Induced Infertile Mouse.

About 50% of infertility is caused by men. This study aimed to investigate the efficiency of photobiomodulation on spermatogenesis in a busulfan-induced infertile mouse as a testicular degeneration treatment. Thirty-two adult NMRI male mice were divided into 4 groups: control, busulfan, PBMT 0.03 J/cm2, and laser 0.2 J/cm2. In the study, azoospermia was induced by busulfan as a testicular degeneration, and then, they were treated using photobiomodulation therapy at 0.03 J/cm2 and 0.2 J/cm2 energy densities. Sperm parameters, stereological analysis, serum testosterone levels, together with SDH activity, MDA production oxidized as a marker for lipid peroxidation, glutathione (GSSG) and glutathione (GSH), mitochondrial membrane permeability (MMP), reactive oxygen species (ROS) production, and ATP production as well as TUNEL assay were assessed. Photobiomodulation therapy with 0.03 J/cm2 energy densities group revealed a significant increase the testosterone hormone level and spermatogenic cells with the reduction of apoptotic cells and marked increase in GSH, ATP, and SDH levels and decrease the levels of MDA and ROS production in the busulfan-induced mice when compared with the control and sham groups. In conclusion, the photobiomodulation therapy (0.03 J/cm2 energy density) may provide benefits on the spermatogenesis following busulfan injection and might be an alternative treatment to the patients with oligospermia and azoospermia in a dose-dependent manner.

Amniotic fluid-derived exosomes improved spermatogenesis in a rat model of azoospermia.

AIMS: This study aimed to explore the therapeutic effects of amniotic fluid-derived extracellular vesicles including exosomes (AF-Exos) on the recovery of sperm production capacity in a rat model of azoospermia. MAIN METHODS: The non-obstructive azoospermia (NOA) was induced in rats using intratesticular administration of Busulfan. Azoospermia was confirmed by testis histology. AF-Exos samples containing 10 or 40 µg exosomal proteins were injected into testicular tissue of NOA rats. After two months, the recovery of spermatogenesis was monitored via histopathological staining, spermiogram, and hormonal analysis. Immunohistochemistry staining for OCT-3/4 was used to identify of spermatogonial progenitors. The expression of DAZL and VASA, was also measured. KEY FINDINGS: AF-Exos exhibited sphere-shaped morphology with the mean diameter and zeta potential of 50 ± 7.521 nm and -7.16 mV. Immunoblots revealed that isolated nanoparticles were CD63, CD9, and CD81 positive. Histopathological evaluation revealed that spermatogenesis was improved significantly in NOA rats after AF-Exos injection. Data showed that the sperm parameters and spermatogenesis index were significantly improved after AF-Exos injection compared to azoospermic groups. OCT-3/4+ cells were increased in NOA rats after AF-Exos injection, showing the restoration of spermatogenesis. In the present study, both doses of exosome (10 and 40 µg) restored the testicular function of NOA rats. DAZL and VASA were increased significantly in animals who received 40 µg exosomal protein compared to azoospermic rats. Except in a high dose of AF-Exos (40 µg) for Testosterone and FSH, no statistically significant differences were found regarding hormones post-exosome injection. SIGNIFICANCE: Our study demonstrated that AF-Exos regenerated spermatogenesis and improved sperm quality in NOA rats.

The marker of alkyl DNA base damage, N7-methylguanine, is associated with semen quality in men.

Sperm DNA contains a range of DNA base damage that can arise, in part, from exposure to methylating agents. However, the effects are not fully characterized and so the aim of this study was to investigate associations between semen quality and the levels of N7-methyldeoxyguanosine (N7-MedG), a marker of exposure to methylating agents, and other markers of DNA damage and DNA methylation. Sperm samples were collected from 105 men attending an assisted reproduction clinic as part of a couple undergoing treatment for infertility and semen quality assessed manually according to WHO guidelines. Semen levels of N7-MedG, quantified by immunoslotblot, were significantly higher in men with sperm concentration < 15 × 106/ml (p ≤ 0.01), semen volume < 1.5 ml (p ≤ 0.05) and also in men with any aspect of semen quality below WHO reference levels (p ≤ 0.001). Measures of neutral Comet DNA damage were correlated with semen quality in a univariate analysis but not after adjustment for N7-MedG levels. Sperm concentration was negatively associated with % methylation at the gene for DAZL but no other marker of global or gene-specific DNA methylation. Results support the hypothesis that the known toxic and DNA damaging properties of alkylating agent exposure may have direct deleterious consequences on semen quality.