Optimization of HPV DNA detection in urine by improving collection, storage, and extraction.

The benefits of using urine for the detection of human papillomavirus (HPV) DNA have been evaluated in disease surveillance, epidemiological studies, and screening for cervical cancers in specific subgroups. HPV DNA testing in urine is being considered for important purposes, notably the monitoring of HPV vaccination in adolescent girls and young women who do not wish to have a vaginal examination. The need to optimize and standardize sampling, storage, and processing has been reported.In this paper, we examined the impact of a DNA-conservation buffer, the extraction method, and urine sampling on the detection of HPV DNA and human DNA in urine provided by 44 women with a cytologically normal but HPV DNA-positive cervical sample. Ten women provided first-void and midstream urine samples. DNA analysis was performed using real-time PCR to allow quantification of HPV and human DNA.The results showed that an optimized method for HPV DNA detection in urine should (a) prevent DNA degradation during extraction and storage, (b) recover cell-free HPV DNA in addition to cell-associated DNA, (c) process a sufficient volume of urine, and (d) use a first-void sample.In addition, we found that detectable human DNA in urine may not be a good internal control for sample validity. HPV prevalence data that are based on urine samples collected, stored, and/or processed under suboptimal conditions may underestimate infection rates.

Toxicity of the antitumoral drug datelliptium in hepatic cells: Use of models in vitro for the prediction of toxicity in vivo.

Datelliptium is a DNA-intercalating agent derived from ellipticine. The drug has potent antitumoral activity in vitro and in vivo. The first clinical use of the drug revealed unexpected hepatotoxic effects in humans that had not been observed in animals. Using different hepatic models in vitro (rat hepatocytes in suspension and in culture, cultured human hepatocytes and rat and human hepatoma cell lines), the possibility of prediction in vitro of the hepatic toxicity of a drug has been investigated. Cytotoxic effects were evaluated by measuring the leakage of intracellular lactate dehydrogenase and the ability of cells to reduce MTT after exposure to concentrations of datelliptium ranging from 0.1 to 1000 mum. According to these endpoint parameters, the concentrations of the drug that produced 50% of maximal inhibitory effect (IC(50)) were in the range 100 to 195 mum in rat hepatocyte suspension and hepatocyte cultures after 2 hr of treatment, 7-9 mum in cultured rat and human hepatocytes after 23 hr of treatment, and about 200-320 mum in HepG2 and FaO cells after 23 hr of treatment. Metabolic parameters were generally more sensitive than cytotoxic endpoints for detecting toxic effects of datelliptium on hepatocytes in the two experimental models used. Metabolic effects on rat hepatocyte suspension and culture were evaluated respectively after 2 and 23 hr of exposure to the drug. Triglyceride secretion was the most sensitive parameter and the IC(10) values (concentration causing 10% of maximal inhibitory effect) were 0.03 and 0.9 mum in hepatocyte suspension and culture, respectively. Glycogen, albumin and cellular protein synthesis were similarly altered in both cellular systems and the IC(10) values were in the range 0.5-3.5 mum. Ureogenesis and gluconeogenesis were relatively insensitive parameters in cell suspensions (IC(10) values 16.4 and 10.3 mum, respectively) compared with those in hepatocyte culture (IC(10) values 3.6 and 3.1 mum, respectively). The concentrations of datelliptium reported in blood, and particularly in liver, are higher than the concentrations that produce impairment of cell metabolism in vitro. This may be an indicator of the toxicological risk of datelliptium and anticipates the hepatotoxicity observed in vivo.

Adult rat liver slices as a model for studying the hepatotoxicity of vincaalkaloids.

There are certain disadvantages associated with the use of isolated or cultured cells including the need to use proteolytic enzymes for their isolation and loss of tissue organization. In order to provide an in vitro system for toxicological studies that preserves tissue integrity, a method for preparation and incubation of adult rat liver slices has been developed. Fresh ultra-thin liver slices were produced in large quantities at a rapid rate under conditions that cause minimal tissue trauma. They were incubated in a system that has been designed to allow optimum gas and nutrient diffusion. Several biochemical parameters (lactate dehydrogenase (LDH) leakage, ATP content, protein synthesis and secretion) were monitored during a 20-hr incubation period. This liver slice model was used to study the toxicity of four vincaalkaloids: vincristine, vindesine, vinblastine and navelbine. Treatment with the vincaalkaloids resulted in an inhibition of protein synthesis and secretion without any effect on LDH leakage.