The use of genomics technology to investigate gene expression changes in cultured human liver cells.

The field of genomics has great potential in toxicology; however, the technology is still in its infancy and there are many questions that need to be addressed. In this study we focus on the use of toxicogenomics for the determination of gene expression changes associated with hepatotoxicity. The human hepatoma cell line HepG2 was used to assess the toxic effects of two well-studied hepatotoxins, carbon tetrachloride (CCl(4)) and ethanol (EtOH). Replicate dishes of HepG2 cells were exposed to two concentrations of CCl(4) and EtOH--doses which caused 20% and 50% cell death (as determined by the MTT assay) were chosen [0.18% and 0.4% (v/v) CCl(4); 2.5% and 5% (v/v) EtOH] and the cells exposed for periods of 2 and 24 h. mRNA was extracted and used to probe Atlas Human Toxicology II arrays (Clontech). Preliminary data revealed that following a 2-h exposure at the low doses of both compounds, few changes in gene expression were detected. However, after 24-h exposure of the cells to the same low concentration of both compounds, multiple changes in gene expression were observed, many of which were specific to the individual hepatotoxins, presumably reflecting their different mechanisms of action. CCl(4) treatment of HepG2 cells gave rise to treatment specific up-regulation of genes involved in extracellular transport and cell signalling, whereas EtOH treatment gave rise predominantly to down-regulation of genes involved in stress response and metabolism. In addition, changes in regulation of certain genes (involved in stress response and cell cycle) were common to both treatments. Exposure of HepG2 cells to higher doses of the hepatotoxins gave rise to more changes in gene expression at lower exposure times. These results strongly suggest that different mechanisms of hepatotoxicity may be associated with specific patterns of gene expression, while some genes associated with common cellular responses may be useful as early markers of toxicity.

N of 1 trials. Practical tools for medication management.

BACKGROUND: In single patient (n of 1) trials, a patient acts as his or her own control in a study comparing the effectiveness of a drug with placebo or another drug. The aim of a single patient trial (SPT) is to identify the best treatment for the individual patient, formalising a 'trial of treatment' by using blinding and multiple crossover periods. OBJECTIVE: We have successfully piloted SPTs for osteoarthritis and attention deficit hyperactivity disorder, and several others are currently being piloted. Barriers encountered include: obtaining identical placebos, ethical approval for individual SPTs, standardising doses, determining length of treatment periods, patient withdrawals and cost of the SPTs. DISCUSSION: Only prescribing medications if an individual has been shown to be a responder can greatly benefit general practitioners, patients and the healthcare system. We have established the infrastructure necessary to offer a single patient trial service to GPs and patients anywhere in Australia. This has the potential to revolutionise prescribing for certain chronic conditions.

Preliminary experiences with a single-patient trials service in general practice.

OBJECTIVE: To pilot a single-patient trials (SPTs) service in general practice, designed to improve decision-making about long-term medications for chronic conditions. DESIGN: 12-week within-patient, randomised, double-blind, placebo-controlled, crossover comparison of ibuprofen with paracetamol for osteoarthritis, involving three pairs of two-week treatment periods for each participating patient. SETTING AND PATIENTS: Patients attending an academic general practice with a clinical diagnosis of osteoarthritis, with pain of at least a month's duration severe enough to warrant consideration of long-term non-steroidal anti-inflammatory drug (NSAID) use. MAIN OUTCOME MEASURES: Pain and stiffness; measures of overall arthritis compared with previous fortnight; preference for NSAID at the end of each two-week treatment period; use of escape analgesia; side effects; and management changes as a result of the SPTs. RESULTS: Eight of 14 patients completed SPTs. One was a clear responder to NSAIDs, five were non-responders, and two were indefinite. Of the five who were using NSAIDs' before the SPT, two continued and three ceased using them. Clinically useful information assisted decision-making for all eight participants. Medication management changed for six. CONCLUSIONS: Single-patient trials can be successfully implemented in general practice and might be a valuable method for GPs to identify patients who respond to medication for chronic stable conditions such as osteoarthritis, in which individual response to medication is variable.

Managing ADHD in general practice. N of 1 trials can help!

OBJECTIVE: To pilot single patient trials designed to improve decision making about stimulant use for attention deficit hyperactivity disorder (ADHD) in general practice. METHOD: Patients previously stabilised on dexamphetamine were enrolled from a general practice. Each undertook a six week same patient randomised, double blind, placebo controlled crossover comparison of dexamphetamine with placebo for ADHD. Rating scales were completed weekly by self, parent and teacher. RESULTS: Three of the four patients were clear responders to dexamphetamine (including a noncompleter, as his results still demonstrated a clear response). The results were clinically useful in each case. Management was confirmed for three patients and changed for one (who ceased dexamphetamine). DISCUSSION: Prescribing stimulant medications only to children with diagnosed ADHD and who are found to respond, limits use of these worrisome drugs to those who will respond, and minimises their use in those who will not benefit.

High levels of cathepsin B predict poor outcome in patients with breast cancer.

Cathepsin B (CB) is a thiol-stimulated protease implicated in cancer invasion and metastasis. Other proteases involved in cancer spread such as urokinase-type plasminogen activator (uPA) and cathepsin D have previously been shown to be prognostic markers in breast cancer. CB was assayed by ELISA in 193 patients with primary breast cancer. CB levels were significantly higher in both primary and metastatic breast tumors than in fibroadenomas (p = 0.0001). In the primary carcinomas, CB levels showed no significant correlation with either nodal status, tumor size or estrogen receptor (ER) status. Patients with primary breast cancers containing high levels of CB had a significantly shorter disease-free interval (p = 0.01, chi-square = 6.61) and overall survival (p = 0.014, chi-square = 6.08) than patients with low levels of the protease. However, in multivariate analysis, using nodal status, tumor size, ER status and urokinase plasminogen activator (uPA), CB was not an independent prognostic marker. In contrast, nodal status, ER status and uPA were prognostic in multivariate analysis. In conclusion, CB, like certain other proteases implicated in cancer metastasis, correlates with poor outcome in patients with breast cancer. These results thus support the evidence from model systems linking CB to cancer spread. Inhibition of CB expression or activity might therefore be exploited for anti-metastatic therapies.