Rapamycin prevents cyclophosphamide-induced ovarian follicular loss and potentially inhibits tumour proliferation in a breast cancer xenograft mouse model.

STUDY QUESTION: To what extent and via what mechanism does the concomitant administration of rapamycin (a follicle activation pathway inhibitor and antitumour agent) and cyclophosphamide (a highly toxic ovarian anticancer agent) prevent cyclophosphamide-induced ovarian reserve loss and inhibit tumour proliferation in a breast cancer xenograft mouse model? SUMMARY ANSWER: Daily concomitant administration of rapamycin and a cyclic regimen of cyclophosphamide, which has sufficient antitumour effects as a single agent, suppressed cyclophosphamide-induced primordial follicle loss by inhibiting primordial follicle activation in a breast cancer xenograft mouse model, suggesting the potential of an additive inhibitory effect against tumour proliferation. WHAT IS KNOWN ALREADY: Cyclophosphamide stimulates primordial follicles by activating the mammalian target of the rapamycin (mTOR) pathway, resulting in the accumulation of primary follicles, most of which undergo apoptosis. Rapamycin, an mTOR inhibitor, regulates primordial follicle activation and exhibits potential inhibitory effects against breast cancer cell proliferation. STUDY DESIGN, SIZE, DURATION: To assess ovarian follicular apoptosis, 3 weeks after administering breast cancer cells, 8-week-old mice were randomized into three treatment groups: control, cyclophosphamide, and cyclophosphamide + rapamycin (Cy + Rap) (n = 5 or 6 mice/group). Mice were treated with rapamycin or vehicle control for 1 week, followed by a single dose of cyclophosphamide or vehicle control. Subsequently, the ovaries were resected 24 h after cyclophosphamide administration (short-term treatment groups). To evaluate follicle abundance and the mTOR pathway in ovaries, as well as the antitumour effects and impact on the mTOR pathway in tumours, 8-week-old xenograft breast cancer transplanted mice were randomized into three treatment groups: vehicle control, Cy, and Cy + Rap (n = 6 or 7 mice/group). Rapamycin (5 mg/kg) or the vehicle was administered daily for 29 days. Cyclophosphamide (120 mg/kg) or the vehicle was administered thrice weekly (long-term treatment groups). The tumour diameter was measured weekly. Seven days after the last cyclophosphamide treatment, the ovaries were harvested, fixed, and sectioned (for follicle counting) or frozen (for further analysis). Similarly, the tumours were resected and fixed or frozen. PARTICIPANTS/MATERIALS, SETTING, METHODS: Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was performed to examine ovarian follicular apoptosis in the short-term treatment groups. All subsequent experiments were conducted in the long-term treatment groups. Tumour growth was evaluated using the tumour volume index. The tumour volume index indicates the relative volume, compared to the volume 3 weeks after tumour cell injection (at treatment initiation) set to 100%. Tumour cell proliferation was evaluated by Ki-67 immunostaining. Activation of the mTOR pathway in tumours was assessed using the protein extracts from tumours and analysed by western blotting. Haematoxylin and eosin staining of ovaries was used to perform differential follicle counts for primordial, primary, secondary, antral, and atretic follicles. Activation of the mTOR pathway in ovaries was assessed using protein extracts from whole ovaries and analysed by western blotting. Localization of mTOR pathway activation within ovaries was assessed by performing anti-phospho-S6 kinase (downstream of mTOR pathway) immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Ovaries of the short-term treatment groups were resected 24 h after cyclophosphamide administration and subjected to TUNEL staining of apoptotic cells. No TUNEL-positive primordial follicles were detected in the control, Cy, and Cy + Rap groups. Conversely, many granulosa cells of growing follicles were TUNEL positive in the Cy group but negative in the control and Cy + Rap groups. All subsequent experimental results were obtained from the long-term treatment groups. The tumour volume index stabilized at a mean of 160-200% in the Cy group and 130% in the Cy + Rap group throughout the treatment period. In contrast, tumours in the vehicle control group grew continuously with a mean tumour volume index of 600%, significantly greater than that of the two treatment groups. Based on the western blot analysis of tumours, the mTOR pathway was activated in the vehicle control group and downregulated in the Cy + Rap group when compared with the control and Cy groups. Ki-67 immunostaining of tumours showed significant inhibition of cell proliferation in the Cy + Rap group when compared with that in the control and Cy groups. The ovarian follicle count revealed that the Cy group had significantly fewer primordial follicles (P < 0.001) than the control group, whereas the Cy + Rap group had significantly higher number of primordial follicles (P < 0.001, 2.5 times) than the Cy group. The ratio of primary to primordial follicles was twice as high in the Cy group than in the control group; however, no significant difference was observed between the control group and the Cy + Rap group. Western blot analysis of ovaries revealed that the mTOR pathway was activated by cyclophosphamide and inhibited by rapamycin. The phospho-S6 kinase (pS6K)-positive primordial follicle rate was 2.7 times higher in the Cy group than in the control group. However, this effect was suppressed to a level similar to the control group in the Cy + Rap group. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The combinatorial treatment of breast cancer tumours with rapamycin and cyclophosphamide elicited inhibitory effects on cell proliferative potential compared to cyclophosphamide monotherapy. However, no statistically significant additive effect was observed on tumour volume. Thus, the beneficial antitumour effect afforded by rapamycin administration on breast cancer could not be definitively proven. Although rapamycin has ovarian-protective effects, it does not fully counteract the ovarian toxicity of cyclophosphamide. Nevertheless, rapamycin is advantageous as an ovarian protective agent as it can be used in combination with other ovarian protective agents, such as hormonal therapy. Hence, in combination with other agents, mTOR inhibitors may be sufficiently ovario-protective against high-dose and cyclic cyclophosphamide regimens. WIDER IMPLICATIONS OF THE FINDINGS: Compared with a cyclic cyclophosphamide regimen that replicates human clinical practice under breast cancer-bearing conditions, the combination with rapamycin mitigates the ovarian follicle loss of cyclophosphamide without interfering with the anticipated antitumour effects. Hence, rapamycin may represent a new non-invasive treatment option for cyclophosphamide-induced ovarian dysfunction in breast cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This work was not financially supported. The authors declare that they have no conflict of interest.

Antioxidant vitamins and lysophospholipids are critical for inducing mouse spermatogenesis under organ culture conditions.

In vitro mouse spermatogenesis using a classical organ culture method became possible by supplementing basal culture medium with only the product of bovine serum albumin purified by chromatography (AlbuMAX), which indicated that AlbuMAX contained every chemical factor necessary for mouse spermatogenesis. However, since the identity of these factors was unclear, improvements in culture media and our understanding of the nutritional and signal substances required for spermatogenesis were hindered. In the present study, chemically defined media (CDM) without AlbuMAX was used to evaluate each supplementary factor and their combinations for the induction of spermatogenesis. Similar to in vivo conditions, retinoic acid, triiodothyronine (T3 ), and testosterone (T) were needed. Based on differences in spermatogenic competence between AlbuMAX, fetal bovine serum, and adult bovine serum, we identified α-tocopherol, which strongly promoted spermatogenesis when combined with ascorbic acid and glutathione. Differences were also observed in the abilities of lipids extracted from AlbuMAX using two different methods to induce spermatogenesis. This led to the identification of lysophospholipids, particularly lysophosphatidylcholine, lysophosphatidic acid, and lysophosphatidylserine, as important molecules for spermatogenesis. New CDM formulated based on these results induced and promoted spermatogenesis as efficiently as AlbuMAX-containing medium. In vitro spermatogenesis with CDM may provide a unique experimental system for research on spermatogenesis that cannot be performed in in vivo experiments.

[A Case of Lymphoepithelioma-Like Carcinoma of the Ureter].

Lymphoepithelioma-like carcinoma (LELC) of the ureter is very rare and only 14 previous cases have been reported. Here, we report a case of LELC of the ureter. A 76-year-old woman was admitted to our hospital complaining of gross hematuria. Left ureteral cancer was suspected by the imaging examination, and laparoscopic left total nephroureterectomy was performed. Histopathological examination showed pure type of LELC in the ureter. She is alive without disease recurrence at fifteen months after surgery.

Transcriptional regulator Bhlhe40 works as a cofactor of T-bet in the regulation of IFN-γ production in iNKT cells.

Invariant natural killer T (iNKT) cells are a subset of innate-like T cells that act as important mediators of immune responses. In particular, iNKT cells have the ability to immediately produce large amounts of IFN-γ upon activation and thus initiate immune responses in various pathological conditions. However, molecular mechanisms that control IFN-γ production in iNKT cells are not fully understood. Here, we report that basic helix-loop-helix transcription factor family, member e40 (Bhlhe40), is an important regulator for IFN-γ production in iNKT cells. Bhlhe40 is highly expressed in stage 3 thymic iNKT cells and iNKT1 subsets, and the level of Bhlhe40 mRNA expression is correlated with Ifng mRNA expression in the resting state. Although Bhlhe40-deficient mice show normal iNKT cell development, Bhlhe40-deficient iNKT cells show significant impairment of IFN-γ production and antitumor effects. Bhlhe40 alone shows no significant effects on Ifng promoter activities but contributes to enhance T-box transcription factor Tbx21 (T-bet)-mediated Ifng promoter activation. Chromatin immunoprecipitation analysis revealed that Bhlhe40 accumulates in the T-box region of the Ifng locus and contributes to histone H3-lysine 9 acetylation of the Ifng locus, which is impaired without T-bet conditions. These results indicate that Bhlhe40 works as a cofactor of T-bet for enhancing IFN-γ production in iNKT cells.

In vitro spermatogenesis in two-dimensionally spread mouse testis tissues.

PURPOSE: Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this condition, however, a tissue piece tends to round up to be compact, whose central region suffers from shortage of nutrients and oxygen. In this study, the authors improved the culture condition by spreading each tissue thin and flat, by which they were able to get better access to the oxygen and nutrients. METHODS: Immature mouse testis tissues placed on agarose gel block were forced to spread flat by covering with a polydimethylsiloxane (PDMS) ceiling chip (PC chip). They were then cultured for weeks and evaluated by the transgene expression of Acr-Gfp, which reflects the progression of spermatogenesis. RESULTS: Testis tissues covered with PC chip initiated and maintained spermatogenesis in its wider region than those without PC chip covering. Flow cytometric analysis demonstrated that the PC method yielded more numerous meiotic germ cells than those without PC. Immunohistochemical examination confirmed the authentic histological figure of spermatogenesis from spermatogonia up to round or elongating spermatids. CONCLUSIONS: The PC chip method is simple and effective to improve the efficiency of in vitro spermatogenesis in the organ culture system.

A monolayer microfluidic device supporting mouse spermatogenesis with improved visibility.

In our previous study, we produced a microfluidic device (MFD) which successfully maintained spermatogenesis for over 6 months in mouse testis tissues loaded in the device. In the present study, we developed a new MFD, a monolayer device (ML-D) with a barrier structure consisting of pillars and slits, which is simpler in design and easier to make. This ML-D was also effective for inducing mouse spermatogenesis and maintained it for a longer period than the conventional culture method. In addition, we devised a way of introducing sample tissue into the device during its production, just before bonding the upper layer of polydimethylsiloxane (PDMS) and bottom glass slide. The tissue can obtain nutrients horizontally from the medium running beside it and oxygen vertically from above through PDMS. In addition, the glass slide set at the bottom improved the visibility of the sample tissue with an inverted microscope. When we took photos of cultured tissue of the Acr-Gfp transgenic mouse testis in ML-D sequentially every day, morphological changes of the acrosome during spermiogenesis were successfully recorded. The ML-D is simple in design and useful for culturing testis tissue for inducing and maintaining spermatogenesis with clearer visibility. Due to the new method of sample loading, tissues other than testis should also be applicable.

Neonatal testis growth recreated in vitro by two-dimensional organ spreading.

Organ culture experiments can be hampered by central degeneration or necrosis due to the inadequate permeation of oxygen and nutrients, which deteriorates the function and growth of cultured tissues. In the current study, we aimed to overcome this limitation of organ culture through spreading the tissue two dimensionally on an agarose gel stand and molding into a disc shape by placing a ceiling of polydimethylsiloxane (PDMS) chip, which is highly oxygen permeable. By this, every part of the tissue can receive a sufficient supply of oxygen through PDMS as well as nutrients through the agarose gel below. This method not only prevented central necrosis of tissues, but also supported the tissue growth over time. In addition, such growth, as volume enlargement, could be easily measured. Under these conditions, we examined the effect of several factors on the growth of neonatal mouse testis, and found that follicle stimulating hormone (FSH) and insulin significantly promoted the growth. These results are in good agreement with previous in vivo reports. Notably, the growth achieved over 7 days in our in vitro system is almost comparable to, about 80% of, that observed in vivo. Thus, we successfully monitored the promotion of tissue growth beyond the limits of the conventional organ culture method. This extremely simple method could offer a unique platform to evaluate the growth as well as functional properties of organs, not only the testis but also others as well.

Comparison of the performance of experienced and novice surgeons: measurement of gripping force during laparoscopic surgery performed on pigs using forceps with pressure sensors.

BACKGROUND: Laparoscopic surgical techniques are difficult to learn, and developing such skills involves a steep learning curve. To ensure surgeons achieve a high skill level, it is important to be able to measure and assess their skills. Therefore, it is necessary to understand the performance differences between experienced and novice surgeons, as such information could be used to help surgeons learn laparoscopic skills. We examined the differences in gripping and reaction force between experienced and novice surgeons during laparoscopic surgery. METHODS: We measured the gripping force generated during laparoscopic surgery performed on pigs using forceps with pressure sensors. Several sensors, including strain gauges, accelerometers, and a potentiometer, were attached to the forceps. This study included 4 experienced and 4 novice surgeons. Each subject was asked to elevate the kidney in order to approach the renal hilus using the forceps. Throughout the experiment, we measured the gripping force and reaction force generated during the movement of the forceps in real time. RESULTS: The experienced and novice surgeons exhibited similar reaction force levels. Conversely, gripping force differed significantly between the groups. The experienced and novice surgeons exhibited mean gripping force levels of 3.06 and 7.15 N, respectively. The gripping force standard deviation values for the experienced and novice surgeons were 1.43 and 3.54 N, respectively. The mean and standard deviation gripping force values of the experienced surgeons were significantly lower than those of the novice surgeons (P = 0.015 and P = 0.011, respectively). CONCLUSIONS: This study indicated that experienced surgeons generate weaker but more stable gripping force than novice surgeons during laparoscopic procedures.

Validation of a patient-specific simulator for laparoscopic renal surgery.

OBJECTIVES: To describe and to validate a novel patient-specific virtual-reality based simulator for laparoscopic surgery. METHODS: Three surgeons carried out 13 preoperative simulations at Yokohama City University Hospital, Yokohama, Kanagawa, Japan, from 2011 to 2012. The procedures included seven nephrectomies, four partial nephrectomies and two pyeloplasties. We evaluated whether the anatomies reproduced by the simulator matched those encountered during the actual operations. Furthermore, the surgeons were asked to use visual analog scales (from 1 to 5; higher scores are better) to evaluate the anatomical integrity and utility of the simulations, and their intraoperative confidence during the subsequent surgical procedures. RESULTS: The simulator reproduced the patients' anatomies almost perfectly. Only a few minor mistakes were identified. Regarding the surgeons' evaluations of the system, the mean scores for the anatomical integrity and utility of the simulations, and the surgeons' intraoperative confidence were 3.4, 4.2 and 4.1, respectively. In all 13 cases, the surgeons were able to carry out preoperative training with ease, and they stated that the simulator was useful for producing preoperative images. CONCLUSIONS: A patient-specific simulator for laparoscopic renal surgery has been successfully developed. This system correctly reproduces anatomical structures, and it seems to be a useful preoperative training tool.

[A Case of Kartagener's Syndrome in Infertile Male Patients].

The patient was a 33-year-old man attending the infertility clinic with primary infertility of 3 years duration. The semen examination showed oligozoospermia and suspected primary male infertility. He had a history of chronic sinusitis and respiratory disease. His chest X-ray showed dextrocardia. Abnormality of the ultrastructure of the cilia of the tract epithelium was found by electron microscopy, and further examination revealed bronchoectasis. We gave him a diagnosis of Kartagener syndrome from these findings. Kartagener syndrome consists of bronchiectasis, sinusitis and situs inversus and is considered a form of primary ciliary dyskinesia (PCD). PCD is also a cause of motor impairment of sperm flagella. This case had successful in-vitro fertilization pregnancy with spermatozoa from the patient.

Prediction of open urinary tract in laparoscopic partial nephrectomy by virtual resection plane visualization.

BACKGROUND: The purpose of this study is presenting a method to predict the presence of an open urinary tract and the position of the opening in laparoscopic partial nephrectomy from three dimensional (3D) computed tomography (CT) images by using novel image segmentation and visualization techniques. METHODS: From CT images of patients who underwent laparoscopic partial nephrectomy, 3D regions of the kidney, urinary tract, and tumor were segmented. For each patient, multiple virtual resection planes of the kidney with different surgical margins (1 mm to 5 mm, every 1 mm) were generated and the presence of an open urinary tract and the position of the opening were predicted from the images. RESULTS: We compared the predictions with actual operations in 5 cases by using recorded video of the operations and operative notes. In terms of the presence of an open urinary tract, agreement of the predictions and the intraoperative results was obtained in all patients. The expected positions of the openings were close to those in the actual operations. CONCLUSIONS: We have developed a method to virtually visualize the resection plane of laparoscopic partial nephrectomy. Image segmentation methods used in this study were precise and effective. The comparison indicated that our method accurately predicted the presence of an open urinary tract and the position of the opening and provided useful preoperative information.

[Clinical investigation of male infertile patients with chromosomal anomalies].

We investigated 470 male patients who came to the Reproduction Medical Center of Yokohama City University Medical Center complaining of infertility between April 2,000 and August 2013. We analyzed the chromosomes of 90 patients whose sperm concentration was below 1.0 × 107/ml. Nineteen of the 90 (21.1%) patients showed sex chromosomal anomalies including 12 Klinefelter syndrome (47, XXY or 46, XY/47, XXY), Robertsonian translocation, 2 autosome-autosome translocation, Y-autosome translocation, 46, X with marker chromosome (46, Xmar⁺), XX male and Y chromosome macrodeletion (46, XYq-). While patients with chromosomal abnormalities except XX male or some of 46, XYq- may succeed in reproduction using testicular sperm extraction-intracytoplasmic sperm injection, we need to inform the patients about the risks of chromosomal abnormalities in the resulting fetus.

[Case of azoospermia patient with a chromosomal abnormality considered a ring Y chromosome].

A 43-year-old man came to our clinic complaining of infertility and semen analysis showed azoospermia. Analysis of chromosomes showed a mosaic 45, XO/46, X, ＋mar1/46, X, ＋mar2 karyotype, and the marker chromosomes were considered to be two kinds of ring Y chromosomes. Y chromosome microdeletion analysis showed partial deletion of Azoospermic Factor (AZF) a, and complete deletion of AZFb and AZFc. The patient gave up having a child because these results indicated that no sperm would be collected even if Testicular Sperm Extraction (TESE) were performed.

Presurgical downstaging of vena caval tumor thrombus in advanced clear cell renal cell carcinoma using temsirolimus.

A 63-year-old man presenting with a 7.2-cm right renal mass, an inferior vena cava tumor thrombus, and pulmonary metastases underwent renal mass biopsy that revealed clear cell renal cell carcinoma. Temsirolimus (25 mg weekly) was given because of the extent of the disease and poor performance status, which resulted in a marked reduction in the tumor thrombus (from level III to level I) after 20 weeks of treatment. Subsequently, radical nephrectomy and tumor thrombectomy were carried out. Final pathological analysis confirmed the diagnosis of high-grade clear cell carcinoma (pT4N0M1). One year after initiation of temsirolimus therapy, the patient remained alive despite the presence of disease.

Dynamic measuring of physical properties for developing a sophisticated preoperative surgical simulator: how much reaction force should a surgical simulator represent to the surgeon?

The acquisition of physical quantities for a living body in surgery is an important and necessary step toward developing a sophisticated preoperative surgical simulator and its validation and navigation. We have developed a multimodal measuring device that minimizes interference with the movements of the surgeon. We conducted nephrectomy surgery using a laboratory animal and successfully acquired physical quantities. From this experiment, we have acquired the following preliminary result. The surgeon feels a gripping force from -3.5 to 4.4N at the handle of the forceps for dissection. We assume that this data is not far from that of a human.

Early ultrasonographic evaluation of tumor thrombus level during sunitinib therapy for renal cell carcinoma.

A 72-year-old man presenting with a 14-cm left renal mass, an inferior vena cava (IVC) tumor thrombus, and pulmonary metastases underwent renal mass biopsy that revealed clear cell renal cell carcinoma. Because of metastases and the extent of the tumor thrombus, sunitinib was administered, which resulted in a marked reduction in the tumor thrombus (from level III to level II after 11 weeks of treatment). Ultrasonography, preceding computed tomography, showed a slight shrinkage of the tumor thrombus level in the first 2 weeks. Therefore, ultrasound may be advantageous to monitor the IVC tumor thrombus level during the early phase of targeted therapy.

Locally advanced endometrial cancer with multiple immune-related adverse events coinciding with the complete response to radiotherapy after immune checkpoint inhibitor therapy: A case report.

We report a case of a 70-year-old female patient with locally advanced endometrial cancer with primary empty sella who developed multiple immune-related adverse events (irAEs), including hypopituitarism coinciding with the complete response to radiotherapy after receiving immune checkpoint inhibitors. A computed tomography scan acquired after a traffic accident led to the discovery of endometrial cancer that had invaded the vulva and primary empty sella. Following adriamycin and cisplatin, pembrolizumab was administered for three cycles. No irAEs were observed during treatment, but the tumor was progressive. The patient underwent radiotherapy for the residual tumor. Four months after the last dose of pembrolizumab, hypopituitarism caused secondary adrenal insufficiency, primary hypothyroidism, and pseudogout at the end of radiotherapy. The tumor later achieved a complete response. In conclusion, radiotherapy after immune checkpoint inhibitor (ICI) therapy is expected to have an antitumor effect by stimulating tumor-specific immunity. However, proper management of irAEs is necessary. Patients with primary empty sella may be prone to pituitary insufficiency induced by ICIs.

Expression, purification, crystallization and preliminary X-ray analysis of 4-hydroxy-3-methyl-2-keto-pentanoate aldolase (asHPAL) from Arthrobacter simplex strain AKU 626.

4-Hydroxy-3-methyl-2-keto-pentanoate aldolase (asHPAL), an enzyme used in the synthesis of (2S,3R,4S)-4-hydroxyisoleucine, was crystallized in the absence and the presence of 2-ketobutyrate as one of its substrates by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. Crystals of asHPAL grown without and with 2-ketobutyrate diffracted to 1.60 and 1.55 Šresolution and belonged to space group C2, with unit-cell parameters a = 116.8, b = 88.2, c = 85.3 Å, ß = 122.3° and a = 116.2, b = 88.1, c = 85.0 Å, ß = 122.3°, respectively.

Evaluation of lens opacity due to inhibition of cholesterol biosynthesis using rat lens explant cultures.

Drug-induced lens opacity has the potential to cause blindness and is of concern in drug development. Inhibition of cholesterol biosynthesis is one of the causes of lens opacity. Lens opacity is only observed after chronic administration in in vivo nonclinical studies in drug development. Thus, to save resources (e.g., time and cost) and to reduce burden on animals, it is required to develop in vitro evaluation systems that can predict and avoid the risk of lens opacity earlier and easier. In this study, we investigated whether rat lens explant cultures could be useful for the evaluation of drug-induced lens opacity via inhibition of cholesterol biosynthesis. Nineteen drugs, including statins, allylamine, thiocarbamate, azole, and morpholine, which inhibit cholesterol biosynthesis, as well as a negative control (acetaminophen, rosiglitazone and troglitazone), were used. Rat lens explants were treated with drugs for 13 days at concentrations close to IC50 values or higher against cholesterol biosynthesis, and lens opacity (severity and region) was evaluated. In most cases, region-specific lens opacity limited in the equator to posterior pole, as observed in vivo was observed at IC50 values or higher concentrations. The severity of opacity was likely to be related to the inhibitory potency toward cholesterol biosynthesis, concentration of drugs distributed in the lens, or time of exposure. Furthermore, GSH levels were also involved in the deterioration of lens opacity. In conclusion, we demonstrated that rat lens explant cultures can be useful to assess the potential drug-induced lens opacity associated with inhibition of cholesterol biosynthesis and to elucidate the mechanisms of lens opacity.