Continuous Toxicity Monitoring Research Articles

Continuous monitoring of toxicity in clinical trials – simulating the risk of stopping prematurely

Continuous monitoring of toxicity in clinical trials – simulating the risk of stopping prematurely

Application of a multi-channel system for continuous monitoring and an early warning system

A multi-channel continuous toxicity monitoring system developed in our laboratory, based on two-stage mini-bioreactors, was successfully implemented in the form of computer-based data acquisition. The multi-channel system consists of a series of a two-stage minibioreactor systems connected by a fiber optic probe to a luminometer, and uses genetically engineered bioluminescent bacteria for the detection of the potential toxicity from the soluble chemicals. This system can be stably and continuously operated due to the separation of the culture reactor from the test reactor and accomplish easy and long-term monitoring without system shut down by abrupt inflows of severe polluting chemicals. Four different recombinant bioluminescent bacteria were used in different channels so that the modes of the samples toxicities can be reasonably identified and evaluated based upon the response signature of each channel. The bioluminescent signatures were delivered from four channels by switching one at once, while the data is automatically logged to an IBM compatible computer. We also achieved the enhancement of the system through the manipulation of the dilution rate and the use of thermo-lux fusion strains. Finally, this system is now being implemented to a drinking water reservoir and river for remote sensing as an early warning system.

Fetal haemoglobin augmentation in E/β0 thalassaemia: clinical and haematological outcome

Patients with E/beta(0) thalassaemia, the most common haemoglobinopathy in many Asian countries, might benefit from drugs that increase fetal and total haemoglobin and thereby decrease the need for transfusions. The long-term clinical efficacy and safety of such therapy is unknown, limiting its use in countries where resources for safe and regular transfusion are scarce. In this study, 45 patients were treated with hydroxyurea (18-20 mg/kg) for 24+/-9 months, hydroxyurea with sodium phenyl butyrate (n=8) and hydroxyurea with erythropoietin (n=9), each for approximately 6 months, and followed for 3 years from study exit. Hydroxyurea had minimal toxicity, resulted in a mean 1.3 g/dl steady-state increase in haemoglobin in 40% of patients, and a milder response (<OR=1 g/dl) in the others. Baseline haemoglobin F was significantly associated with an increase in haemoglobin (P<0.001). Combined treatment with erythropoietin benefited selected patients, but the addition of sodium phenyl butyrate had no effect. Of the 27/45 patients who discontinued regular transfusions before the study, 13 remained transfusion independent during long-term follow-up, 6/13 continued hydroxyurea. Hydroxyurea moderately increased steady-state haemoglobin in a sub-group of E/beta(0) thalassaemia patients and can be considered for patients with intermediate severity disease, thus delaying or avoiding the need for life-long transfusions. Continuous monitoring of toxicity and growth is required.

A Multi-Channel Continuous Water Toxicity Monitoring System: Its Evaluation and Application to Water Discharged from a Power Plant

A multi-channel continuous water toxicity monitoring system was, after confirming the systems' performance, implemented to samples of water discharged from power plants to detect and classify their toxicity using several recombinant bioluminescent bacteria. Each channel of the system is composed of a series of two mini-bioreactors to enable a continuous operation, i.e., without system interruption due to highly toxic samples. A different recombinant bacterial strain was present in each channel: DPD2540 (fabA::lux CDABE), DPD2794 (recA::luxCDABE), and TV1061 (grpE::luxCDABE), which are induced by cell membrane-, DNA-, and protein-damaging agents, respectively. GC2 (lac::luxCDABE) is a constitutive strain, whose bioluminescence is reduced by an increase in cellular toxicity. Phenol and mitomycin C (MMC) were used for evaluating the system's performance to detect toxic chemicals. These samples were injected into the second mini-bioreactor according to a step or bell-curve manner. The field samples used in this study were obtained from the water discharged from two different power plants in Korea--from a nuclear power plant and a thermo-electronic power plant--and were injected into the second mini-bioreactor to initiate the toxicity test. Each channel showed specific bioluminescent (BL) response profiles due to the toxic compounds present in the water samples. Comparing the BL signals between the standard toxic chemical samples and discharged water samples, the equivalent toxicity of the field water could be estimated. Finally, it was proved that this novel continuous toxicity monitoring system can be used as an alternative tool for the quick monitoring and control of water quality, as well as aid in the setting up of a new monitoring strategy to protect the source of tap water and in the prevention of polluted water discharge.

Continuous Toxicity Monitoring in Phase II Trials in Oncology

The goal of a phase II trial in oncology is to evaluate the efficacy of a new therapy. The dose investigated in a phase II trial is usually an estimate of a maximum-tolerated dose obtained in a preceding phase I trial. Because this estimate is imprecise, stopping rules for toxicity are used in many phase II trials. We give recommendations on how to construct stopping rules to monitor toxicity continuously. A table is provided from which Pocock stopping boundaries can be easily obtained for a range of toxicity rates and sample sizes. Estimation of the probability of toxicity and response is also discussed.

Enhancement of the multi-channel continuous monitoring system through the use of Xenorhabdus luminescens lux fusions

The enhancement of the multi-channel continuous toxicity monitoring system developed previously was studied. To achieve better and more stable results from the system, the use of thermo- lux fusion strains that express the luxCDABE genes from Xenorhabdus luminescens was evaluated. A total of six recombinant Escherichia coli strains with the promoters from three oxidative-stress responsive genes, i.e. the katG, sodA and pqi-5 genes, fused to either the lux genes from Vibrio fischeri or X. luminescens were characterized and their responses to different chemicals compared. It was found that the basal level bioluminescence (BL) from the thermo- lux fusion strains was always higher while that of the V. fischeri lux strains were always near or below the lower limit of detection of the system. For example, the katG::V. fischeri lux strain, DPD2511, gave no discernible response due to its low level expression while a fusion of the katG promoter with the X. luminescens lux operon was clearly responsive and capable of detecting hydrogen peroxide down to about 1 ppm. The use of the thermo- lux strains found them to be as sensitive as the V. fischeri lux strains while providing a brighter, more stable basal level bioluminescence, making the analysis and monitoring of water-borne toxicity more reliable.

Surgical management and outcome of tuberculous atlantoaxial dislocation: a 15-year experience.

Tuberculous atlantoaxial dislocation is a rare disease entity. However, tuberculosis continues to be endemic in developing countries. Its earliest clinical presentation may be nonspecific, and delay in diagnosis may lead to irreversible neurological deficit. The management of tuberculous atlantoaxial dislocation includes ventral cervicomedullary decompression, occipitocervical arthrodesis, and administration of antituberculous medications. Eighteen patients with tuberculous atlantoaxial dislocation who presented with neck pain and/or occipital headache, restriction of neck movement, difficulty swallowing, and signs of myelopathy were studied. Four patients had evidence of associated pulmonary tuberculosis. Plain x-rays of the cervical spine, computed tomographic scans, and magnetic resonance images were obtained in all patients for diagnosis and to assess the degree of dislocation and cervicomedullary compression. Simultaneous anterior neural decompression, via a transcervical retropharyngeal approach, and posterior arthrodesis were performed on all patients while they remained under anesthesia. Antituberculous chemotherapy was continued for 18 months. Histopathological analysis of excised tissue was consistent with tuberculosis in all patients. However, Ziehl-Neelsen staining for acid-fast bacilli was positive in two cases, and culture for Mycobacterium tuberculosis was negative in all patients. Patients with severe myelopathy experienced marked improvement. One patient died of fulminant resistant tuberculous meningitis. The transcervical retropharyngeal approach to the craniovertebral junction provides direct access to the lesion and avoids the potential bacterial contamination of the oral and pharyngeal cavity. It also prevents the development of persistent fistulae. Posterior stabilization should be performed directly after anterior neural decompression, while the patient remains under anesthesia, to prevent neurological deterioration before subsequent posterior fixation. This technique also is helpful for early mobilization of patients. The aim of surgical treatment should be to obtain biopsy tissue and to perform radical excision of epidural granulation tissue/abscess and infected bone using microsurgical technique. Antituberculous medication must be continued for 18 months with four drug regimens, and continuous monitoring of drug toxicity should be performed throughout the course of treatment.

Enhancing the sensitivity of a two-stage continuous toxicity monitoring system through the manipulation of the dilution rate

Optimization of the dilution rates has been studied to provide an enhanced sensitivity to toxicity by several recombinant bioluminescent Escherichia coli strains, TV1061 ( grpE::luxCDABE), DPD2794 ( recA::luxCDABE) and DPD2540 ( fabA::luxCDABE), in the two-stage continuous toxicity monitoring system. It was found that the sensitivity of both TV1061 and DPD2794 to a pulse injection of phenol and mitomycin C increased with a decrease in the dilution rate. The sensitivity, however, for all the strains to step injections of the toxic chemicals was found to increase with an increase in the dilution rate up to a certain dilution rate and then decreased, mainly due to the rapid washing out of the injected chemicals. The response kinetics of the strains were explained by evaluating the mode of action of the recombinant bioluminescent bacteria to toxicity with the dilution rate, the operating parameter of minibioreactors under consideration in this study.

A multi-channel continuous toxicity monitoring system using recombinant bioluminescent bacteria for classification of toxicity

A multi-channel system for continuous toxicity monitoring and classification of toxicity was developed based upon a previously developed two-stage minibioreactor system. The multi-channel system consists of a series of a two-stage minibioreactor systems connected by a fiber optic probe to a luminometer. Each channel was used for cultivating different recombinant bacterial strains, such as TV1061 ( grpE::luxCDABE), DPD2794 ( recA::luxCDABE), and DPD2540 ( fabA::luxCDABE), which are induced by protein-, DNA-, and cell membrane damaging-agents, respectively. GC2 ( lac::luxCDABE) is a bacterium expressing bioluminescence constitutively, which shows a reduction in its light level as cellular toxicity increases. Artificial wastewater samples were made by combining toxic chemicals, including Mitomycin C (a representative DNA damaging agent), phenol (a representative protein damaging agent), and cerulenin (a representative cell membrane damaging agent), and injecting this sample into each channel in order to simulate the detection of toxicity for mixed chemical samples. Each channel showed a specific bioluminescent response due to the toxic chemicals contained in the sample wastewater, while GC2 showed a general response to cellular toxicity. By using this multi-channel continuous toxicity monitoring system, classification of toxicity in field samples was found to be possible.

Optimal continuous sequential boundaries for monitoring toxicity in clinical trials: a restricted search algorithm.

Continuous monitoring of severe adverse experiences can ensure the timely termination of a clinical trial if the therapy is shown to be harmful. In this paper we present methods for choosing a stopping rule for continuous monitoring of toxicity in small trials. They are especially useful for small phase II trials of about 30 patients for monitoring a binary toxicity event that is observed relatively quickly compared to the efficacy outcome. In 1987 Goldman described an algorithm for computing the exact type I error rate (alpha) and power (1-beta) of a specified discrete stopping boundary for sequential monitoring of a study with a fixed maximum number of patients (N) to be enrolled on the experimental therapy. Only an upper boundary was used since trials are only terminated for an excess frequency of toxicity and not for a low rate. By repeated use of this algorithm a stopping rule can be identified which has nearly the chosen level of (alpha) and a reasonable power depending on the design parameters of the study. The work reported here embeds this earlier algorithm as a subroutine in a larger FORTRAN program which searches all boundaries that fulfil constraints on size and power, as specified by the user. The search is restricted so that only those boundaries with size in a small neighbourhood of the chosen alpha are examined and displayed if the power is above a set minimum. These restrictions reduce the number of boundaries examined to only 0.4 per cent of all possible boundaries, thus reducing running time to a practical few seconds. Many such boundaries exist, the one with the largest power can then be chosen for monitoring the trial. The average sample number (ASN) and the expected relative loss (ERL) are also computed. The criterion for choosing may also be based on small ASN or low ERL in addition to power and appropriate alpha.

A two-stage minibioreactor system for continuous toxicity monitoring

A two-stage minibioreactor system was successfully developed for continuous toxicity monitoring. This system consists of two minibioreactors in series. Recombinant Escherichia coli DPD2794 containing a RecA::luxCDABE fusion as a model strain was utilized to monitor environmental insults to DNA, with mitomycin C as a model toxicant. Pulse type exposures were used to evaluate the system’s reproducibility and reliability. Step inputs of mitomycin C have been adopted to show the system’s stability. The system’s ability to monitor the possible upsets or accidental discharges of toxic chemicals was also evaluated with these step insults. All the data demonstrated that this two-stage minibioreactor system using recombinant bacteria containing stress promoters fused with lux genes is quite appropriate for continuous toxicity monitoring. Long-term operation and minimized media-usage have been investigated. Thus application to many different areas, including an early warning system of wastewater biotreatment plant upsets and the monitoring and tracking of accidental spills, discharges or failures in plant operation are plausible.

Bioluminescent Reporter Bacterium for Toxicity Monitoring in Biological Wastewater Treatment Systems

Toxic shock due to certain chemical loads in biological wastewater treatment systems can result in death of microorganisms and loss of floc structure. To overcome the limitations of existing approaches to toxicity monitoring, genes encoding enzymes for light production were inserted to a bacterium (Shk 1) isolated from activated sludge. The Shk 1 bioreporter indicated a toxic response to concentrations of cadmium (0.01 to 10 000 mg/L), 2,4‐dinitrophenol (0.01 to 1 000 mg/L), and hydroquinone (0.01 to 10 000 mg/L) by reductions in initial levels of bioluminescence on exposure to the toxicant. The decrease in bioluminescence was more severe with increasing toxicant concentration. Bioluminescence did not decrease in response to ethanol concentrations up to 10 000 mg/L or to pH conditions between 6.1 and 7.9. A continuous toxicity monitoring system using this bioreporter was developed for influent wastewater and tested with hydroquinone. The reporter exhibited a rapid and proportional decrease in bioluminescence in response to increasing hydroquinone concentrations.