3D printing: Innovative solutions for patients and pharmaceutical industry.

Three-dimensional (3D) printing is an emerging technology with great potential in pharmaceutical applications, providing innovative solutions for both patients and pharmaceutical industry. This technology offers precise construction of the structure of dosage forms and can benefit drug product design by providing versatile release modes to meet clinical needs and facilitating patient-centric treatment, such as personalized dosing, accommodate treatment of specific disease states or patient populations. Utilization of 3D printing also facilitates digital drug product development and manufacturing. Development of 3D printing at early clinical stages and commercial scale pharmaceutical manufacturing has substantially advanced in recent years. In this review, we discuss how 3D printing accelerates early-stage drug development, including pre-clinical research and early phase human studies, and facilitates late-stage product manufacturing as well as how the technology can benefit patients. The advantages, current status, and challenges of employing 3D printing in large scale manufacturing and personalized dosing are introduced respectively. The considerations and efforts of regulatory agencies to address 3D printing technology are also discussed.

Melt extrusion deposition (MED™) 3D printing technology - A paradigm shift in design and development of modified release drug products.

Three-dimensional printing (3DP) technology offers unique advantages for pharmaceutical applications. However, most of current 3D printing methods and instrumentations are not specifically designed and developed for pharmaceutical applications. To meet the needs in pharmaceutical applications for precision, compatibility with a wide range of pharmaceutical excipients and drug materials without additional processing, high throughput and GMP compliance, an extrusion-based 3D printer based on Melt Extrusion Deposition (MED™) 3D printing technology was developed in this study. This technology can process powder pharmaceutical excipients and drugs directly without the need of preparing filament as required by FDM 3D printing. Six different tablet designs based on compartment models were used to demonstrate the precision and reproducibility of this technology. The designed tablets were fabricated using the GMP-compliant MED™ 3D printer and were evaluated in vitro for drug release and in vivo for selected designs using male beagle dogs. Tablet designs with one or more compartments showed versatile release characteristics in modulating the release onset time, release kinetics, duration of release and mode of release. Multiple drugs or formulations were fabricated into a single tablet to achieve independent release kinetics for each drug or to fine-tune the pharmacokinetic profile of a drug. Building upon the theoretical analysis of models, precision and reproducibility of MED™ 3D printing technology, a novel product development approach, 3D printing formulation by design (3DPFbD®) was developed to provide an efficient tool for fast and efficient pharmaceutical product development. The MED™ 3D printing represents a novel and promising technology platform encompassing design and development of modified drug release products and has potential to impact the drug delivery and pharmaceutical product development.

[Thirdhand smoke: current research status and future prospects].

Thirdhand smoke (THS) is defined as residual tobacco components that remain on indoor surfaces after tobacco has been smoked, such as walls furniture, and dust particles, which are re-emitted into the air. THS also includes secondary pollutants generated from the reaction of surface residual smoke compounds with reactive indoor air pollutants. THS is a new hidden health hazard, with infants and children being most at risk of higher exposure. This article summarized the aging process of secondhand smoke and the mechanism of generation of THS; reviewed the current status of research on THS regarding its chemical constituents, physical and chemical properties, biological toxicity, as well as degree of pollution in China and other countries; and finally provided the perspectives on the future study of THS.

[A investigation of thirdhand smoke pollution in 3 types of places of Nanjing, 2014].

OBJECTIVE: To investigate thirdhand smoke (THS) pollution in certain places of Nanjing, as well as to analyze its distribution characteristics. METHODS: From March to May, 2014, we selected 3 types of places (residencies, public places and transportation vehicles) that were close to people's living in Jianye,Yuhua,Jiangning,Xuanwu,Gulou and Pukou districts of Nanjing city.For each of the above 3 types of places, 2-3 smoking and non-smoking (smoking ban) locations were investigated, totally 51 locations, 9-10 samples were collected each location, totally 477 samples. The surface wipe sampling method in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilized to quantify the levels of nicotine that served as the tracer of THS pollution.One-way ANOVA and t-tests were employed to compare the levels of nicotine collected at different places and locations. RESULTS: Totally 477 samples were collected in this study, of which 27.0% was from residencies (129/477), 61.0% (291/477) from public places and 11.9% (57/477) from transportations. The levels of indoor surface nicotine in smoking residences, public places and transportations were (214 ± 55),(1 408 ± 177) and (1 511 ± 785) µg/m(2), respectively, which were all higher than those in the corresponding non-smoking places ((23 ± 9),(62 ± 11), and (46 ± 15) µg/m(2); t values were 13.79, 13.15, 3.45, respectively. P values were <0.001, <0.001 and 0.006, respectively).In the smoking places, the levels of surface nicotine on walls, desks, sofas, cabinets, door backsides and air conditioning openings were (171 ± 62),(232 ± 38),(373 ± 151),(903 ± 239), (978 ± 212), (1 721 ± 517) µg/m(2) (F = 7.06, P = 0.009).In the smoking condition, the levels of surface nicotine collected from public places were higher (F = 9.25, P = 0.024), while under non-smoking (smoking ban) conditions, the levels of surface nicotine collected from residences were lower (F = 7.88, P < 0.001). CONCLUSION: THS pollution was widespread in public places, residences and transportations in Nanjing city, which was more serious in the smoking environments than non-smoking (smoking ban) environments; the contamination was less serious in non-smoking (smoking ban) private residences; in the smoking condition, the levels of surface nicotine were relatively high at locations close to air conditioning openings, door backsides and cabinets.

3,3'-Diindolylmethane exhibits antileukemic activity in vitro and in vivo through a Akt-dependent process.

3,3'-Diindolylmethane (DIM), one of the active products derived from Brassica plants, is a promising antitumor agent. The present study indicated that DIM significantly induced apoptosis in U937 human leukemia cells in dose- and time-dependent manners. These events were also noted in other human leukemia cells (Jurkat and HL-60) and primary human leukemia cells (AML) but not in normal bone marrow mononuclear cells. We also found that DIM-induced lethality is associated with caspases activation, myeloid cell leukemia-1 (Mcl-1) down-regulation, p21(cip1/waf1) up-regulation, and Akt inactivation accompanied by c-jun NH2-terminal kinase (JNK) activation. Enforced activation of Akt by a constitutively active Akt construct prevented DIM-mediated caspase activation, Mcl-1 down-regulation, JNK activation, and apoptosis. Conversely, DIM lethality was potentiated by the PI3K inhibitor LY294002. Interruption of the JNK pathway by pharmacologic or genetic approaches attenuated DIM-induced caspases activation, Mcl-1 down-regulation, and apoptosis. Lastly, DIM inhibits tumor growth of mouse U937 xenograft, which was related to induction of apoptosis and inactivation of Akt, as well as activation of JNK. Collectively, these findings suggest that DIM induces apoptosis in human leukemia cell lines and primary human leukemia cells, and exhibits antileukemic activity in vivo through Akt inactivation and JNK activation.

Ursolic acid induces apoptosis in human leukaemia cells and exhibits anti-leukaemic activity in nude mice through the PKB pathway.

BACKGROUND AND PURPOSE: Ursolic acid (UA) has been extensively used as an anti-leukaemic agent in traditional Chinese medicine. In the present study, we investigated the ability of UA to induce apoptosis in human leukaemia cells in relation to its effects on caspase activation, Mcl-1 down-regulation and perturbations in stress-induced signalling pathways such as PKB and JNK. EXPERIMENTAL APPROACH: Leukaemia cells were treated with UA after which apoptosis, caspase activation, PKB and JNK signalling pathways were evaluated. The anti-tumour activity of UA was evaluated using xenograft mouse model. KEY RESULTS: UA induced apoptosis in human leukaemia cells in a dose- and time-dependent manner; this was associated with caspase activation, down-regulation of Mcl-1 and inactivation of PKB accompanied by activation of JNK. Enforced activation of PKB by a constitutively active PKB construct prevented UA-mediated JNK activation, Mcl-1 down-regulation, caspase activation and apoptosis. Conversely, UA lethality was potentiated by the PI3-kinase inhibitor LY294002. Interruption of the JNK pathway by pharmacological or genetic (e.g. siRNA) attenuated UA-induced apoptosis. Furthermore, UA-mediated inhibition of tumour growth in vivo was associated with induction of apoptosis, inactivation of PKB as well as activation of JNK. CONCLUSIONS AND IMPLICATIONS: Collectively, these findings suggest a hierarchical model of UA-induced apoptosis in human leukaemia cells in which UA induces PKB inactivation, leading to JNK activation and culminating in Mcl-1 down-regulation, caspase activation and apoptosis. These findings indicate that interruption of PKB/JNK pathways may represent a novel therapeutic strategy in haematological malignancies.

Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in vivo.

In this study, we investigated the functional role of Akt and c-jun-NH(2)-kinase (JNK) signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 downregulation, cytochrome c release from mitochondria, and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspase activation, and apoptosis. Conversely, LY294002 and a dominant-negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of the JNK pathway showed marked reduction in apigenin-induced caspase activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied by inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens.

Protein dynamics via computational microscope.

The purpose of this overview is to provide a concise introduction to the methodology and current advances in molecular dynamics (MD) simulations. MD simulations emerged as a powerful and popular tool to study dynamic behavior of proteins and macromolecule complexes at the atomic resolution. This approach can extend static structural data, such as X-ray crystallography, into dynamic domains with realistic timescales (up to millisecond) and high precision, therefore becoming a veritable computational microscope. This perspective covers current advances and methodology in the simulation of protein folding and drug design as illustrated by several important published examples. Overall, recent progress in the simulation field points to the direction that MD will have significant impact on molecular biology and pharmaceutical science.

Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/ß-catenin pathway in human colorectal adenocarcinoma DLD1 cells.

Long term exposure to arsenic can increase incidence of human cancers, such as skin, lung, and colon rectum. The mechanism of arsenic induced carcinogenesis is still unclear. It is generally believed that reactive oxygen species (ROS) may play an important role in this process. In the present study, we investigate the possible linkage between ROS, ß-catenin and arsenic induced transformation and tumorigenesis in human colorectal adenocarcinoma cell line, DLD1 cells. Our results show that arsenic was able to activate p47(phox) and p67(phox), two key proteins for activation of NADPH oxidase. Arsenic was also able to generate ROS in DLD1 cells. Arsenic increased ß-catenin expression level and its promoter activity. ROS played a major role in arsenic-induced ß-catenin activation. Treatment of DLD1 cells by arsenic enhanced both transformation and tumorigenesis of these cells. The tumor volumes of arsenic treated group were much larger than those without arsenic treatment. Addition of either superoxide dismutase (SOD) or catalase reduced arsenic induced cell transformation and tumor formation. The results indicate that ROS are involved in arsenic induced cell transformation and tumor formation possible through Wnt/ß-catenin pathway in human colorectal adenocarcinoma cell line DLD1 cells.

Cadmium induces autophagy through ROS-dependent activation of the LKB1-AMPK signaling in skin epidermal cells.

Cadmium is a toxic heavy metal which is environmentally and occupationally relevant. The mechanisms underlying cadmium-induced autophagy are not yet completely understood. The present study shows that cadmium induces autophagy, as demonstrated by the increase of LC3-II formation and the GFP-LC3 puncta cells. The induction of autophagosomes was directly visualized by electron microscopy in cadmium-exposed skin epidermal cells. Blockage of LKB1 or AMPK by siRNA transfection suppressed cadmium-induced autophagy. Cadmium-induced autophagy was inhibited in dominant-negative AMPK-transfected cells, whereas it was accelerated in cells transfected with the constitutively active form of AMPK. mTOR signaling, a negative regulator of autophagy, was downregulated in cadmium-exposed cells. In addition, cadmium generated reactive oxygen species (ROS) at relatively low levels, and caused poly(ADP-ribose) polymerase-1 (PARP) activation and ATP depletion. Inhibition of PARP by pharmacological inhibitors or its siRNA transfection suppressed ATP reduction and autophagy in cadmium-exposed cells. Furthermore, cadmium-induced autophagy signaling was attenuated by either exogenous addition of catalase and superoxide dismutase, or by overexpression of these enzymes. Consequently, these results suggest that cadmium-mediated ROS generation causes PARP activation and energy depletion, and eventually induces autophagy through the activation of LKB1-AMPK signaling and the down-regulation of mTOR in skin epidermal cells.

Nickel-induced down-regulation of ΔNp63 and its role in the proliferation of keratinocytes.

Epidemiological, animal, and cell studies have demonstrated that nickel compounds are human carcinogens. The mechanisms of their carcinogenic actions remain to be investigated. p63, a close homologue of the p53 tumor suppressor protein, has been linked to cell fate determination and/or maintenance of self-renewing populations in several epithelial tissues, including skin, mammary gland, and prostate. ΔNp63, a dominant negative isoform of p63, is amplified in a variety of epithelial tumors including squamous cell carcinomas and carcinomas of the prostate and mammary glands. The present study shows that nickel suppressed ΔNp63 expression in a short-time treatment (up to 48 h). Nickel treatment caused activation of NF-κB. Blockage of NF-κB partially reversed nickel-induced ΔNp63 suppression. Nickel decreased interferon regulatory factor (IRF) 3 and IRF7, IKKε, and Sp100. Over-expression of IRF3 increased ΔNp63 expression suppressed by nickel. Nickel was able to activate p21, and its activation was offset by the over-expression of ΔNp63. In turn, elevated p63 expression counteracted the ability of nickel to restrict cell growth. The present study demonstrated that nickel decreased interferon regulatory proteins IRF3 and IRF7, and activated NF-κB, resulting in ΔNp63 suppression and then p21 up-regulation. ΔNp63 plays an important role in nickel-induced cell proliferation.

Interruption of the MEK/ERK signaling cascade promotes dihydroartemisinin-induced apoptosis in vitro and in vivo.

Artemisinin, the active principle of the Chinese medicinal herb Artemisia annua, and its derivatives (i.e. dihydroartemisinin, DHA) were reported to exhibit anti-tumor activity both in vitro and in vivo. The purpose of the present study was to investigate the functional role of Mitogen-Activated Protein Kinase (MEK)/Extracellular signal-regulated protein Kinase (ERK) signaling cascade in dihydroartemisinin (DHA)-induced apoptosis in human leukemia cells in vitro and anti-leukemic activity in vivo. Human leukemia cells were treated with DHA in dose- and time-dependent manners, after which apoptosis, caspase activation, Mcl-1 expression, and cell signaling pathways were evaluated. Parallel studies were performed in AML and ALL primary human leukemia cells. In vivo anti-leukemic activity mediated by DHA was also investigated using U937 xenograft mouse model. Exposure of DHA resulted in a pronounced increase in apoptosis in both transformed and primary human leukemia cells but not in normal peripheral blood mononuclear cells. DHA-induced apoptosis was accompanied by caspase activation, cytochrome c release, Mcl-1 down-regulation, as well as MEK/ERK inactivation. Pretreatment with MEK inhibitor PD98059, which potentiated DHA-mediated MEK and ERK inactivation, intensified DHA-mediated apoptosis. Conversely, enforced expression of a constitutively active MEK1 attenuated DHA-induced apoptosis. Furthermore, DHA-mediated inhibition of tumor growth of mouse U937 xenograft was associated with induction of apoptosis and inactivation of ERK. The findings in the present study showed that DHA-induced apoptosis in human leukemia cells in vitro and exhibited an anti-leukemic activity in vivo through a process that involves MEK/ERK inactivation, Mcl-1 down-regulation, culminating in cytochrome c release and caspase activation.

The dual roles of c-Jun NH2-terminal kinase signaling in Cr(VI)-induced apoptosis in JB6 cells.

Occupational exposure to chromium (Cr) compounds has been shown to cause serious toxic and carcinogenic effects. The skin is an important target for the compounds in industrially exposed Cr workers. c-Jun NH(2)-terminal kinase (JNK) regulates cell proliferation, apoptosis, and differentiation. This protein's effects on cellular response depend upon the cell type and stimuli. The mechanisms by which hexavalent chromium (Cr(VI)) leads to apoptosis in the skin are unclear at present. The aim of this study is to examine whether JNK regulates apoptosis in Cr(VI)-exposed mouse JB6 epidermal cells. The present study showed that Cr(VI) induced apoptotic cell death through JNK activation. The blockage of JNK by small interference RNA (si-RNA) transfection suppressed Cr(VI)-induced apoptotic cell death with the concomitant downregulation of antiapoptotic Bcl-2 family proteins, mitochondrial membrane depolarization (Δψm), caspase activation, and poly (ADP-ribose) polymerase cleavage. However, inhibition of c-Jun expression by si-RNA transfection enhanced cytotoxicity, which corresponded to increasing apoptosis and Δψm. This phenomenon is associated with p53 activation caused by increasing reactive oxygen species (ROS) levels because of the downregulation of superoxide dismutase expression in si-c-Jun-transfected cells. Taken together, Cr(VI) induces apoptosis via JNK-mediated signaling, whereas c-Jun activation acts as an inhibitor of apoptotic signaling. Additionally, ROS generated by Cr(VI) is a pivotal regulator of JNK.

Quercetin induces tumor-selective apoptosis through downregulation of Mcl-1 and activation of Bax.

PURPOSE: To investigate the in vivo antitumor efficacy of quercetin in U937 xenografts and the functional roles of Mcl-1 and Bax in quercetin-induced apoptosis in human leukemia. EXPERIMENTAL DESIGN: Leukemia cells were treated with quercetin, after which apoptosis, Mcl-1 expression, and Bax activation and translocation were evaluated. The efficacy of quercetin as well as Mcl-1 expression and Bax activation were investigated in xenografts of U937 cells. RESULTS: Administration of quercetin caused pronounced apoptosis in both transformed and primary leukemia cells but not in normal blood peripheral mononuclear cells. Quercetin-induced apoptosis was accompanied by Mcl-1 downregulation and Bax conformational change and mitochondrial translocation that triggered cytochrome c release. Knockdown of Bax by siRNA reversed quercetin-induced apoptosis and abrogated the activation of caspase and apoptosis. Ectopic expression of Mcl-1 attenuated quercetin-mediated Bax activation, translocation, and cell death. Conversely, interruption of Mcl-1 by siRNA enhanced Bax activation and translocation, as well as lethality induced by quercetin. However, the absence of Bax had no effect on quercetin-mediated Mcl-1 downregulation. Furthermore, in vivo administration of quercetin attenuated tumor growth in U937 xenografts. The TUNEL-positive apoptotic cells in tumor sections increased in quercetin-treated mice as compared with controls. Mcl-1 downregulation and Bax activation were also observed in xenografts. CONCLUSIONS: These data suggest that quercetin may be useful for the treatment of leukemia by preferentially inducing apoptosis in leukemia versus normal hematopoietic cells through a process involving Mcl-1 downregulation, which, in turn, potentiates Bax activation and mitochondrial translocation, culminating in apoptosis.

Induction of apoptosis in human leukemia cells by grape seed extract occurs via activation of c-Jun NH2-terminal kinase.

PURPOSE: To characterize the functional role of c-Jun NH(2)-terminal kinase (JNK) and other apoptotic pathways in grape seed extract (GSE)-induced apoptosis in human leukemia cells by using pharmacologic and genetic approaches. EXPERIMENTAL DESIGN: Jurkat cells were treated with various concentrations of GSE for 12 and 24 h or with 50 microg/mL GSE for various time intervals, after which apoptosis, caspase activation, and cell signaling pathways were evaluated. Parallel studies were done in U937 and HL-60 human leukemia cells. RESULTS: Exposure of Jurkat cells to GSE resulted in dose- and time-dependent increase in apoptosis and caspase activation, events associated with the pronounced increase in Cip1/p21 protein level. Furthermore, treatment of Jurkat cells with GSE resulted in marked increase in levels of phospho-JNK. Conversely, interruption of the JNK pathway by pharmacologic inhibitor (e.g., SP600125) or genetic (e.g., small interfering RNA) approaches displayed significant protection against GSE-mediated lethality in Jurkat cells. CONCLUSIONS: The result of the present study showed that GSE induces apoptosis in Jurkat cells through a process that involves sustained JNK activation and Cip1/p21 up-regulation, culminating in caspase activation.

Effects of trichlorfon on progesterone production in cultured human granulosa-lutein cells.

Trichlorfon, a widely used organophophorus pesticide, has been reported to disrupt reproductive function in human and animal. However, the mechanisms have not been clearly elucidated. In the present study, the effects of trichlorfon on the biosynthesis of progesterone in the primary human granulosa-lutein cells (hGLCs) and the related pathway were investigated. Results showed that progesterone production in hGLCs treated with trichlorfon decreased significantly while cell viability was not affected. Trichlorfon also inhibited FSH-stimulated progesterone production, and this inhibition could not be reversed by 8-bromo-cAMP. However, trichlorfon did not affect the intracellular cAMP contents in the basal and FSH-stimulated conditions. These results suggested that the site in the steroid biosynthesis pathway affected by trichlorfon occurred downstream of PKA activation in hGLCs. Furthermore, our results found that 22(R)-hydroxycholesterol (22R-HC) could remove the inhibitory action of trichlorfon on progesterone biosynthesis, indicating that trichlorfon caused a disruption of cholesterol transport across mitochondrial membranes, which was further confirmed by the observation that trichlorfon dose-dependently inhibited the mRNA level of the steroidogenic acute regulatory protein (StAR). These results suggested that trichlorfon inhibited steroidogenesis in hGLCs by reducing StAR gene expression, which may further contribute to the pathogenesis of trichlorfon-induced reproductive dysfunction.

Elevated prostaglandin E2 level via cPLA2--COX-2--mPGES-1 pathway involved in bladder carcinogenesis induced by terephthalic acid-calculi in Wistar rats.

To investigate the prostaglandin E2 (PGE2) biosynthetic mechanism in bladder carcinogenesis, we established Wistar rat model of bladder papilloma and transitional cell carcinoma (TCC) induced by 5% terephthalic acid (TPA) treatment. Then, the mRNA level of cytosolic phospholipase A2 (cPLA2), cyclooxygenases (COX)-1 and -2, membrane-bound PGE2 synthases (mPGES)-1 and -2 was detected using reverse transcription polymerase chain reaction (RT-PCR). Immunoblotting was applied to detect the expression of COX-2 protein. Proliferating cell nuclear antigen (PCNA) was determined by immunohistochemistry. In addition, the level of PGE2 was measured by radioimmunoassay (RIA). Bladder papilloma (100%, 8/8) was examined in rats after 24-week treatment, and bladder TCC (80%, 16/20) was found after 48-week treatment. Histopathological changes were not found in control group rats. The incidence of bladder papilloma and TCC in test group was significantly higher than that in control group (P<0.01). The mRNA levels of cPLA2, COX-2 and mPGES-1 in the bladder papilloma and TCC were significantly higher than those in normal bladder (P<0.01), while the mRNA levels of COX-1 and mPGES-2 in TCC were unchanged compared with normal bladder. Bladder TCC exhibited a substantial expression of COX-2 protein. On the contrary, normal bladder tissue barely expresses COX-2 protein. PCNA labeling index (LI) and the level of PGE2 in bladder papilloma are much higher than those in normal bladder (P<0.01), but lower than those in bladder TCC (P<0.05). In conclusion, increasing PGE2 level via cPLA2--COX-2--mPGES-1 pathway may play an important role in rat bladder carcinogenesis. PGE2 may be a biomarker for the development of bladder TCC. cPLA2 and mPGES-1 may be targets for development of novel chemoprevention strategies for bladder TCC.

Carbaryl inhibits basal and FSH-induced progesterone biosynthesis of primary human granulosa-lutein cells.

Carbaryl is known to impede female reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into the mechanisms, this study was conducted to raise fresh concerns about the potential effects of carbaryl on steroidogenesis by primary human granulosa-lutein cells (hGLCs) and explore the possible nature of this action. hGLCs were co-incubated with various concentrations of carbaryl at 0, 1, 5, 25, 125 micromol/L for 24 h to examine effects of this carbamate pesticide on progesterone accumulation. We observed that the carbaryl inhibited basal and FSH-induced progesterone production in a dose-dependent manner. We also investigated the effects of carbaryl on 22(R)-hydroxycholesterol (22R-HC)-stimulated progesterone yield, basal and FSH-stimulated StAR gene expression and cyclic adenosine monophosphate (cAMP) production, as well as forskolin (non-specific activator of adenylyl cyclase)-induced progesterone and cAMP production of hGLCs. We found that the decreased progesterone biosynthesis was accompanied with a reduced cAMP abundance on both basal and FSH-induced condition. Furthermore, our results demonstrated that the 22R-HC could remove the carbaryl-induced restraint of progesterone biosynthesis, suggesting that carbaryl caused a disruption of cholesterol transport across mitochondrial membranes, which was further confirmed by the observation that carbaryl inhibited the gene expression of steroidogenic acute regulatory protein (StAR). In addition, the inhibitory effects of carbaryl on progesterone and cAMP production were completely reversed by addition of forskolin to the cell culture, which indicated a repaired site on the upstream components of adenylate cyclase or adenylate cyclase per se by carbaryl in the cAMP-mediated signal pathway. All the effects mentioned above were not due to a detrimental action of carbaryl on cell viability by MTS assay. In conclusion, carbaryl may inhibit steroidogenesis, at least in part, by obstructing the delivery of cholesterol over mitochondrial membranes and attenuating cAMP generation.

Genotoxic effects on spermatozoa of carbaryl-exposed workers.

Carbaryl, one of the most important insecticides, is widely produced and used. To explore carbaryl-induced genotoxic effects of spermatozoa, particularly DNA damage and chromosome aberrations (CA), we first examined conventional semen parameters, the progression and motion parameters of the spermatozoa among 16 carbaryl-exposed workers and 30 internal and external control individuals. Sperm DNA damage represented as positive percentage of DNA fragmentation was detected by a modified terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Then numerical CA of chromosome X, Y, and 18 were investigated by multicolor fluorescence in situ hybridization (FISH). The results showed significant differences in the percentage of sperm abnormality between carbaryl-exposed group and the external control group (p = 0.008). Mean (+/-SD) percentage of spermatozoa with fragmented DNA in carbaryl-exposed group (21.04 +/- 8.88%) was significantly higher than those in the internal (13.36 +/- 12.17%) and external control groups (13.92 +/- 7.15%), respectively (p = 0.035 and p = 0.030). Using FISH, we observed the frequency of sperm sex chromosome disomy was 0.661 +/- 0.238% in the exposed group, which was significantly higher than that in the external control group (0.386 +/- 0.140%) (p = 0.001), and the carbaryl-exposed group (0.276 +/- 0.126%) had an elevated chromosome 18 disomy compared with the internal (0.195 +/- 0.094%) and external control groups (0.124 +/- 0.068%), respectively (p < 0.05 and p < 0.01). In addition, carbaryl-exposed donors had significantly higher sperm nullisomic frequencies of sex chromosomes and chromosome 18 than the external controls (p < 0.01) but not the internal controls. In summary, the frequencies of aneuploidy and numerical CA showed significant differences between exposed group and control groups (p < 0.05 and/or p < 0.01). Moreover, positive correlations were found between sex chromosome disomy, aneuploidy rate, and morphologic abnormalities in spermatozoa of all donors (r = 0.564 and r = 0.555, p < 0.01). Our findings suggested that carbaryl might induce morphologic abnormalities and genotoxic defects of spermatozoa among exposed workers by causing DNA fragmentation and numerical CA in spermatogenesis as a potential genotoxicant. The evidence also indicated that the spermatotoxicity induced by carbaryl exposure might be related to adverse reproductive outcomes.