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Primary testicular cell coculture model has been used to evaluate testicular abnormalities during development, and was able to identify the testicular toxicity of phthalates. However, the primary testicular cell coculture model has disadvantages in employing animals for the isolation of testicular cells, and the complicated isolation procedure leads to inconsistent results. We developed an invitro testicular coculture model from rodent testicular cell lines, including spermatogonial cells, Sertoli cells, and Leydig cells with specified cell density and extracellular matrix (ECM) composition. Using comparative high-content analysis of F-actin cytoskeletal structure between the coculture and single cell culture models, we demonstrated a 3D structure of the coculture, which created an invivo-like niche, and maintained and supported germ cells within a 3D environment. We validated this model by discriminating between reproductive toxicants and nontoxicants among 32 compounds in comparison to the single cell culture models. Furthermore, we conducted a comparison between the invitro (IC50) and invivo reproductive toxicity testing (lowest observed adverse effect level on reproductive system). We found the invitro coculture model could classify the tested compounds into 4 clusters, and identify the most toxic reproductive substances, with high concordance, sensitivity, and specificity of 84%, 86.21%, and 100%, respectively. We observed a strong correlation of IC50 between the invitro coculture model and the invivo testing results. Our results suggest that this novel invitro coculture model may be useful for screening testicular toxicants and prioritize chemicals for further assessment in the future.

Bisphenol A (BPA), an endocrine-disrupting compound, was found to be a testicular toxicant in animal models. Bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) were recently introduced to the market as alternatives to BPA. However, toxicological data of these compounds in the male reproductive system are still limited so far. This study developed and validated an automated multi-parametric high-content analysis (HCA) using the C18-4 spermatogonial cell line as a model. We applied these validated HCA, including nuclear morphology, DNA content, cell cycle progression, DNA synthesis, cytoskeleton integrity, and DNA damage responses, to characterize and compare the testicular toxicities of BPA and 3 selected commercial available BPA analogues, BPS, BPAF, and TBBPA. HCA revealed BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS, including dose- and time-dependent alterations in nuclear morphology, cell cycle, DNA damage responses, and perturbation of the cytoskeleton. Our results demonstrated that this specific culture model together with HCA can be utilized for quantitative screening and discriminating of chemical-specific testicular toxicity in spermatogonial cells. It also provides a fast and cost-effective approach for the identification of environmental chemicals that could have detrimental effects on reproduction.

A unique noncontact single cell manipulation technique based on the actuation of magnetic nanorods (MNRs) or clusters (MCs) by nonuniform alternating magnetic fields (nuAMFs) is demonstrated. Compared to the actuation of MNRs/MCs by conventional magnetophoresis, the motion of MNRs/MCs actuated by nuAMFs can be tuned by additional parameters including the shape of MNRs/MCs and the frequency of the applied magnetic fields. The manipulation of a single cell by an actuated MNR/MC are divided into five stages, i.e., approaching, pushing, carrying, dragging, and releasing. The interactions between the MNR/MC and the cell in these stages are investigated in detail both experimentally and numerically. Other applications of cell manipulation, such as concentrating cells at target locations and accumulating MNRs/MCs onto a single cell, are also demonstrated. The single cell manipulation system is simple, low-cost, and low-power consumption, and helps advance the state-of-the-art of single-particle manipulation.

1-Bromopropane (1-BP), a halogenated alkane, was introduced into the workplace as an alternative to ozone-layer depleting solvents (ODA) after the discovery of the reproductive and hematopoietic toxicities of 2-brompropane (2-BP) in workers (Kim et al., 1997; Yu et al., 1999a, c, 2001a). 2-BP was first used as an ODA in Korea and Japan and discovered to cause reproductive disorders in workers (Kim et al., 1997; Park et al., 1997). Animal studies confirmed its adverse effects, which included reductions in testes weight and sperm counts, atrophy of the seminiferous tubules, a decrease in the number of ovarian follicles and an increase in irregular estrous cycles (Ichihara et al., 1997, 1999, 2000a,b, 2004a, 2005; Kamijima et al., 1997a, b; Nakajima et al., 1997a, 1997b; Wang et al., 2002, 2003; Yu et al., 1999a, b, 2001a, b; Yamada et al., 2003). The isomer 1-BP was thereafter introduced and approved as an ODA by Environmental Protection Agency’s (EPA, 2007). Subsequent animal studies identified the potential for 1-BP-mediated reproductive and neurotoxicity (Yu et al., 1998a, 2001a). 1-BP is categorized as a high-production volume chemical (Eisenberg and Ramsey, 2010). Its usage has increased dramatically, rising to around 20 million pounds/year, which could result in a widespread occupational exposure (Anderson et al., 2010; NTP, 2013; OSHA, 1999). 1-BP is demonstrated to be a potent neurotoxic compound compared with 2-BP. In addition, 1-BP has reproductive toxicity, but the target cells are different from those of 2-BP. For example, exposure to 1-BP inhibits spermiation in male rats and disrupts the development of follicles in female rats, in contrast to 2-BP, which targets spermatogonia and oocytes in primordial follicles. Because the first animal study revealing the neurotoxicity of 1-BP (Yu et al., 1998a), over a dozen human cases of neurotoxicity have been reported with manifestations of 1-BP toxicity. Among 1-BP exposed workers, dose-dependent prolongation of distal latency in the tibial nerve with decreased vibratory sensation in the lower extremities has been shown (Ichihara et al., 2004b). Species and strain-specific effects of 1-BP were observed in rats and mice (Ichihara et al., 2012; Liu et al., 2009). The majority of toxicological studies were conducted in rats. In a comparative study between the 2 inbred strains of rats, F344 and Wistar, neurotoxicity revealed by distal latency was more significant in F344 than in Wistar rats. Furthermore, mice were reported to be more susceptible than rats to 1-BP-mediated hepatotoxicity and reproductive toxicity (Liu et al., 2009). Hepatotoxicity and male reproductive toxicity were compared among the 3 strains of mice (C57BL/6J, DBA/2J, and BALB/cA) exposed to 1-BP at 0, 50, 110, and 250 ppm for 8 h/day for 28 days by inhalation. Histopathological evaluation of the liver damage showed a significantly larger area of necrosis and more degenerative lobules in BALB/cA in the order of BALB/cA > C57BL/6J > DBA/2J. BALB/cA showed higher CYP2E1 protein level and lower total glutathione (GSH) content and glutathione-S-transferases (GST) activity in the liver than DBA/2J. These results indicate that BALB/cA mice are the most susceptible to the hepatotoxicity of 1-BP among the 3 strains tested, and that CYP2E1 and GSH level/GST activity may contribute to the susceptibility to 1-BP hepatotoxicity. Exposure to ≥ 50 ppm of 1-BP also decreased sperm count and sperm motility and increased sperms with abnormal heads in all 3 strain mice, whereas rats exposed to 200 ppm of 1-BP for 12 weeks showed no changes in sperm count and sperm motility (Ichihara et al., 2000b), suggesting mice are far more susceptible than rats. Most recently, a long-term inhalation animal study from the National Toxicological Program (NTP) found that exposure of male and female F344/N rats to 1-BP significantly increased the incidences of adenomas of the large intestine and skin neoplasms (Morgan et al., 2011; NTP, 2013). In male rats, the incidence of malignant mesothelioma was statistically significantly increased at 500 ppm. There was no evidence of carcinogenic activity of 1-BP in male B6C3F1 mice; however, significantly increased incidences of alveolar/bronchiolar neoplasms of the lung were present in female mice. The mechanism of the different incidence of the carcinogenesis observed between rat and mice and male and female is still unclear, which makes it uncertain how to translate these results from animals to humans. Proposed Occupational Exposure limits (OELs) for 1-BP are diverse in both the selection of critical effects and judgments of remaining uncertainty because its toxicological mechanisms are still poorly known. The OEL values differ by ∼10-fold. Toxicology Excellence for Risk Assessment evaluated the underlying basis of existing OELs through critical effect, benchmark dose, and uncertainty factor, and concluded that the critical effect has decreased the live litter size with a benchmark dose lower confidence limit of 190 ppm (Maier et al., 2004). The OEL of 20 ppm is derived using an uncertainty factor of 10-fold, which is composed of 3-fold for extrapolation from an experimental animal study to humans for expected toxicodynamic differences and 3-fold for expected human variability in toxicokinetics and toxicodynamics within the worker population. The American Conference of Industrial Hygienists (ACGIH) recommends an 8-h time weighted average threshold limit values of 0.10 ppm to provide protection against the potential for neurotoxicity, hepatotoxicity, and reproductive and developmental toxicity in 1-BP exposed workers (ACGIH, 2014). Other professional organizations and manufacturers have recommended exposure limits (RELs) ranging from 20 to 100 ppm (Stelljes and Wood, 2004). The acceptable exposure limit of 1-BP from the U.S. EPA is 25 ppm (8-h time-weighted average). Currently, the National Institute for Occupational Safety and Health does not have a REL, nor does the Occupational Safety and Health Administration have a permissible exposure limit for 1-BP. The potential for human exposure to 1-BP and its adverse effects related with occupation requires understanding of the potential mechanism of these adverse effects in rats and mice as a means of understanding risk in workers. Risk assessment for 1-BP exposure is limited due to lack of animal or human toxicokinetic study. Gas uptake studies analyzed by physiologically based pharmacokinetic (PBPK) models have been used to estimate metabolic parameters for many volatiles (Dobrev et al., 2003, 2008; Lilly et al., 1997). The metabolic constants for a saturable pathway (Vmax and Km) and for a first-order process are typically inferred from the decline in chemical concentration observed in closed chamber exposures. The objective of the current study was to develop a physiologically based PBPK model to simulate the concentration of 1-BP in a closed chamber from a gas-uptake experiment in the F-344 rats. The PBPK model tested the hypotheses, including sex-specific metabolism of 1-BP, and investigated the role of the 2 major metabolic pathways, cytochrome P450 CYP2E1 and GSH conjugation, in the metabolism of 1-BP. The results showed that these 2 metabolic pathways adequately simulated the concentration of 1-BP in the closed chamber. Furthermore, the above model was further tested by simulating the gas-uptake data from a female rat pre-treated with a general P450 suicide inhibitor 1-aminobenzotrizole (ABT) or GSH biosynthesis inhibitor d,l-buthionine (S,R)-sulfoximine (BSO) prior to the exposure of 800 ppm 1-BP. The comparative investigation on the metabolic pathways of 1-BP through the PBPK modeling in both sexes provides critical information in understanding the role of P450 and GSH conjugation in the metabolism of 1-BP and eventually helps to quantitatively extrapolate animal studies to human.

Biological membrane stabilization is essential for maintenance of cellular homeostasis, functionality and appropriate response to various stimuli. Previous studies have showed that accumulation of PKCs in the cell membrane significantly downregulates the membrane fluidity and Ca(2+) influxes through the membranes in activated cells. In addition, membrane-inserted form of PKCs has been found in a variety of resting mammalian cells and tissues. This study is aimed to investigate possible role of the endogenous membrane-associated PKCs in the modulation of basal membrane fluidity. Here, we showed that interfering PKC expression by chronic activation of PKC with phorbol myristate acetate (PMA) or shRNA targeting at PKCα lowered the levels of PKCα in cytosol, peripheral membrane and integral membrane pools, while short-term activation of PKC with PMA induced accumulation of PKCα in the membrane pool accompanied by a dramatic decrease in the cytosol fraction. The lateral membrane mobility increased or decreased in accordance with the abundance alterations in the membrane-associated PKCα by these treatments. In addition, membrane permeability to divalent cations including Ca(2+), Mn(2+) and Ba(2+) were also potentiated or abrogated along with the changes in PKC expression on the plasma membrane. Membrane stabilizer ursodeoxycholate abolished both of the enhanced lateral membrane mobility and permeability to divalent cations due to PKCα deficiency, whereas Gö6983, a PKC antagonist, or Gd(3+) and 2-aminoethyoxydipheyl borne, two Ca(2+) channels blockers, showed no effect, suggesting that this PKC-related regulation is independent of PKC activation or a modulation of specific divalent cation channel. Thus, these data demonstrate that the native membrane-associated PKCα is involved in the maintenance of basal membrane stabilization in resting cells.