Pilot study to detect airborne Mycobacterium tuberculosis exposure in a South African public healthcare facility outpatient clinic.

BACKGROUND: Airborne transmission of Mycobacterium tuberculosis remains an occupational health hazard, particularly in crowded and resource-limited healthcare settings. AIM: To quantify airborne M. tuberculosis in a busy outpatient clinic in Gauteng, South Africa. METHODS: Stationary air samples and samples from healthcare workers (HCWs) were collected in the polyclinic and administrative block. Quantitative real-time polymerase chain reaction (PCR) was used to detect airborne M. tuberculosis. Walkthrough observations and work practices of HCWs were also recorded. FINDINGS: In total, M. tuberculosis was detected in 11 of 49 (22.4%) samples: nine of 25 (36%) HCW samples and two of 24 (8.3%) stationary air samples. Samples from five of 10 medical officers (50%) and three of 13 nurses (23%) were positive. Repeat measurements on different days showed variable results. Most of the HCWs (87.5%) with positive results had been in contact with coughing patients and had not worn respiratory masks despite training. CONCLUSION: The use of air sampling coupled with quantitative real-time PCR is a simple and effective tool to demonstrate the risk of M. tuberculosis exposure. The findings provide an impetus for hospital management to strengthen infection prevention and control measures for tuberculosis.

Noise-induced hearing loss and hearing conservation in the iron and steel industry in South Africa

Background and Purpose: The iron and steel industry in South Africa has been identifi ed as one of the highest risk industries in terms of noise induced hearing loss (NIHL). The National Institute for Occupational Health was commissioned by the Department of Labour to audit the current noise exposure levels and hearing conservation practices in eight major producers of iron and steel; and to make recommendations regarding prevention strategies. Methods: The audit was conducted in two parts: the noise exposure levels and hearing conservation practices were assessed by the occupational hygiene department. The occupational medicine department assessed the hearing conservation policies and procedures; reviewed employees' medical records to ascertain the number of NIHL cases; and conducted verifi cation of audiograms on a sample of employees working in noise zones. Results: Area noise levels exceeding 105 dB(A) were measured in four of the eight workplaces. The estimated average annual incidence of NIHL varied from 0.7 - 8.3/1000/year. All companies did baseline; periodic and exit audiometric testing; but there were notable discrepancies between companies and verifi cation audiograms and differences of more than 20 dB(A) were found. Although information and training on noise was reportedly done in all worksites; a high proportion of workers could not demonstrate correct fi tting of hearing protection devices or recall when last they were trained. Conclusion: A detailed standard operating procedure should be implemented for medical surveillance for NIHL with action timelines that initiate remedial processes prior to employee developing compensable disease. Aggregated audiometric testing results should be communicated to managers and health and safety teams to provide guidance to prioritise areas for control measures. A quality assurance programme for audiometric testing must be implemented. An evaluation tool to measure the effectiveness of the noise and hearing conservation training provided to employees; including contracted employees; should be adopted

Evaluation of performance factors affecting two formulations of cyclosporine in pediatric renal transplant patients.

Success of renal transplantation in children is largely due to improvements in immunosuppressant therapy since the introduction of calcineurin inhibitors. The aim of this study was to identify possible factors that result in formulation differences in the exposure of pediatric patients to cyclosporine (CsA). We examined the handling of the two major formulations of CsA in a group of pediatric renal transplant recipients. The pharmacokinetic profiles of both formulations were assessed, and the data stratified to assess the effects of age, gender, time posttransplant, and other concomitant drug therapy on the two CsA formulations. The microemulsified formulation (MEC) enhanced bioavailability compared to the older oil-based formulation (CYA), especially at C2, with more predictable and consistent absorption in children. This higher bioavailability allowed a 15% reduction of dosing to achieve equal drug exposure. The concentration achieved by MEC at C2 demonstrated a much higher correlation with area under the concentration curve (AUC) than the concentration at C0. In the case of CYA a strong correlation was obtained between AUC and the concentrations obtained at both C0 and C2. Calcium channel blockers increased AUC(0-8) for both CsA formulations. Norfloxacin and pravastatin cotreatment had no effect on either of the CsA formulations. In contrast, the bioavailability of CsA was increased in boys using MEC formulation but this gender-based difference was absent during the use of CYA. This suggests that caution is required for introduction of new formulations of drugs to pediatric patients to evaluate differential effects of age, gender, and concomitant drug therapy.

Effect of lipopolysaccharide (LPS)-evoked host defense activation on hepatic microsomal formation and reduction of sulfamethoxazole hydroxylamine in the rat.

The incidence of adverse reactions to sulfamethoxazole-trimethoprim (SMX-TMP) combination products is higher in patients with AIDS than in the general population. Idiosyncratic adverse reactions to SMX are believed to be dependent upon the formation of the reactive intermediate, sulfamethoxazole hydroxylamine (SMX-HA), and its further oxidation product, nitroso-SMX. Changes in the disposition of SMX have been proposed to contribute to the increased risk of SMX adverse reactions in patients with AIDS. Activation of host defense mechanisms is known to alter drug metabolism and could decrease the enzymatic reduction of SMX-HA to the parent SMX, causing an imbalance in bioactivation and detoxification. We tested this hypothesis in a rat model of lipopolysaccharide (LPS)-evoked host defense activation. Rats were treated i.p. with 1 mg/kg of LPS, and hepatic microsomes were isolated 24 hr after treatment. The bioactivation of SMX to SMX-HA was reduced 50% by pretreatment with LPS (113 +/- 10 vs 65 +/- 4 pmol/min/mg; P < 0.05). However, the NADH-dependent reduction of SMX-HA to SMX was reduced by over 80% (454 +/- 90 vs 81 +/- 48 pmol/min/mg; P < 0.05). A decreased ability to reduce SMX-HA to SMX could predispose patients with systemic activation of host defense mechanisms, such as those with AIDS, to the occurrence of SMX-associated adverse reactions.

Alteration of drug biotransformation and elimination during infection and inflammation.

During infection or inflammation, the expression of cytochrome P450 and its dependent biotransformation pathways are modified. This results in a change in the capacity of the liver to handle drugs and in alterations in the production and elimination of endogenous substances throughout the body. The majority of the CYP isoforms are modified at pre-translational steps in protein synthesis, and, in most cases, cytokines are involved as mediators of the response. Recent information suggests that inflammatory responses that are localized to the CNS cause a loss of CYP within the brain. This is accompanied by a parallel down-regulation of CYP in peripheral organs that is mediated by a signaling pathway between the brain and periphery. This review covers the loss that occurs in the major mammalian CYP families in response to infection/inflammation and the mediator pathways that are key to this response.

Role of cytokines in the lipopolysaccharide-evoked depression of cytochrome P450 in the brain and liver.

A role for cytokines as mediators of the depression in cytochrome P450 activity in brain and liver during CNS inflammation is proposed. Lipopolysaccharide (LPS) was given directly into the lateral ventricle of the brain to mimic a localized CNS infection. CYP1A activity and protein in both brain and liver were depressed in response to this treatment. The administration of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interferon-gamma (IFN-gamma) directly into the lateral ventricle emulated the effects of LPS on CYP1A activity only in the brain. In contrast, these centrally administered cytokines did not produce a concomitant loss of CYP1A activity in the liver. Significant levels of several cytokines (TNF-alpha, IL-1beta, and IFN-gamma) were produced in the serum of animals following intracerebroventricular (i.c.v.) administration of LPS. This production of peripheral cytokines by LPS could not be mimicked by the i.c.v. injection of IL-1beta or TNF-alpha. These results suggest that induction of cytokines in the brain may play a direct role in the depression of CYP1A activity in the CNS following the administration of LPS into the lateral ventricle. The production of cytokines within the brain does not appear to participate in the signaling process in the brain that leads to the concomitant loss of CYP1A activity in the liver. The subsequent production of cytokines in peripheral tissues, however, does appear to play a role in the loss of cytochrome P450 in the liver.

Hepatic and central nervous system cytochrome P450 are down-regulated during lipopolysaccharide-evoked localized inflammation in brain.

The effect of central nervous system inflammation on the levels and activity of hepatic and brain cytochrome P450 were examined in the rat. Brain ethoxyresorufin dealkylkase (EROD) was depressed during localized inflammatory responses evoked by lipopolysaccharide (LPS) injected into the lateral ventricle. This loss was accompanied by a concomitant loss of EROD activity and cytochrome P450 in liver. Similar losses in hepatic enzyme were observed for benzyloxy-resorufin and pentoxy-resorufin dealkylase (CYP2B) and chlorzoxazone hydroxylation (CYP2E). Protein levels of CYP2D and CYP2E1 but not CYP1A also were depressed. Similar i.p. doses of LPS had no effect on hepatic cytochrome P450, indicating that the hepatic effect was not caused by LPS leakage from the central nervous system. Also in support of this contention is that heat shock protein 27 was expressed throughout the brain by LPS given i.c. v. but was undetectable in the liver. Tumor necrosis factor-alpha given i.c.v. depressed EROD activity in the brain but this was not accompanied by a concomitant loss in the liver. Hepatic EROD did respond to the i.p. injection of tumor necrosis factor-alpha. The LPS-evoked loss in hepatic cytochrome P450 could not be prevented by blocking beta-receptor-mediated sympathetic nerve activity. This study demonstrates that localized inflammatory responses in the brain cause a concomitant down-regulation of cytochrome P450 and drug-metabolizing activity in the liver and the brain. The effect on brain cytochrome P450 may be regulated via cytokine-mediated pathways but signaling to the liver does not involve a cytokine-mediated pathway nor a beta-receptor-mediated sympathetic nerve pathway.

Hepatic drug metabolism and immunostimulation.

When host defence mechanisms are stimulated there is a concomitant decrease in cytochrome P450 based drug biotransformation and elimination. This has resulted in a number of clinically important unwanted drug responses in patients with infections or inflammatory responses. The loss in cytochrome P450 is predominantly an effect at the level of the gene expression and the majority of enzyme forms examined to date are involved. Although the effect occurs predominantly in the liver it has been recently shown that inflammatory responses in the brain also cause a loss of the same enzyme forms in that organ. The loss of cytochrome P450 in the brain in response to localised inflammation is accompanied by a similar loss in the liver. The decrease of cytochrome P450 and its dependent drug biotransformation is of concern whenever drugs are used in patients with infections or disease states with an inflammatory component.

Lipopolysaccharide evokes the modulation of brain cytochrome P4501A in the rat.

The cytochrome P450 enzyme system is a multigene family of enzymes that is modulated in the liver during systemic inflammatory responses or during infection Several forms of the enzyme are expressed in discrete areas of the brain and likely play a critical role in the metabolism of drugs and endogenous chemicals in the central nervous system (CNS). Even though the brain responds to inflammation in a manner different from most tissues, we examined the possible modification of a major cytochrome P450 form (CYP1A) in the brain during inflammation confined to that organ. Total brain CYP1A activity, as measured by ethoxyresorufin dealkylase (EROD), was downregulated 24 and 48 h following the administration of a single dose of lipopolysaccharide (LPS). Regionally, a similar effect was determined in the cortex, hippocampus and the mid-brain but the activity in the cerebellum was unaffected. The examination of coronal brain sections using an antibody directed against CYP1A indicated that the enzyme was distributed in discrete cells of the hippocampus, thalamus and cortex and in the tanycytes surrounding the third ventricle. In each of these areas, the immunoreactivity was diminished in animals receiving LPS as compared to saline-treated animals. LPS also evoked the expression of the small molecular weight heat shock protein hsp27 throughout the brain indicating the development of an inflammatory response. These studies indicate that inflammation localized to the CNS causes an alteration in the levels and activity of a major cytochrome P450 form in the brain. This could have implications to the metabolism or activation of drugs and endogenous chemicals in the CNS during a disease state that features an inflammatory component.

Modulation of cytochrome P450 by inflammation in astrocytes.

Activation of systemic host defense mechanisms results in the down-regulation of cytochrome P450 enzymes in the liver. This occurs for various induced and constitutive isoforms of cytochrome P450 in response to cytokines such as IFNs, IL-1, IL-6, and TNF-alpha, which are produced during infection. Although the levels of cytochrome P450 in brain regions are low, the enzymes are regionally distributed and may play a critical role in the activation or degradation of drugs and chemicals in localized areas. If activation of the immune response in the CNS by LPS modulates the activity of cytochrome P450 forms in the brain, this may alter normal metabolic pathways or contribute to drug or chemical toxicity. This hypothesis was addressed by examining the effect of LPS on a major cytochrome P450 form in isolated astrocytes obtained from newborn rats. These cells were shown to express CYP1A1/2 when induced by dibenz[a, h]anthracene (DBA) as determined by enzyme activity, immunohistochemistry, and Western blotting. The treatment of these cells with LPS significantly attenuated the activity of these enzymes but had no effect on CYP1A1/2 protein levels as determined by Western blotting. The lack of effect by detoxified LPS indicated the requirement of the lipid A region on LPS to stimulate this response. Pentoxifylline (PNTX) prevented the LPS evoked decrease in CYP1A1/2 activity suggesting that cytokine release was a required component of this effect in astrocytes. These results indicate that stimulation of the immune response by LPS in isolated astrocytes decreases CYP1A1/2 activity. The release of cytokines is implicated in this effect and thought to participate in the functional inhibition of the enzyme as no effect on CYP1A1/2 protein levels was observed.

Effects of antipyretics on mortality due to influenza B virus in a mouse model of Reye's syndrome.

OBJECTIVES: To determine the effects of acetylsalicylic acid (ASA) and acetaminophen on mortality due to influenza B infection in neonatal and weanling mice, as well as any synergistic, antagonistic or indifferent effects of the combined antipyretic and virus on mortality in mice pretreated with low doses of an industrial surfactant, Toximul MP8, which has been shown to reproduce many of the features of Reye's syndrome. In vitro studies were done to determine whether ASA or acetaminophen altered the normal, interferon-mediated antiviral responses of mammalian cells. The involvement of ASA or other commonly used xenobiotics in the induction of Reye's syndrome following virus illness has not been resolved; to do so, and to elucidate the underlying metabolic mechanism, requires these studies in an animal model. DESIGN: Prospective animal study. ANIMALS: Newborn (945) and weanling (840) Swiss white mice, divided into 12 subgroups. INTERVENTIONS: Some groups received Toximul MP8 before inoculation with a dose of mouse-adapted human influenza B that produces 30% mortality (LD30); after infection, each subgroup received either placebo, ASA or acetaminophen. Mortality counts were taken daily. The in vitro effects of the antipyretics on interferon response were determined using standard virology techniques. OUTCOME MEASURE: Mortality, analyzed by survival curves (log rank test) or cumulative daily mortality (chi 2 analysis). Plaque-reducing dose (PRD50) was used to determine the outcome of the in vitro analyses. RESULTS: In neonatal mice, only subgroups given combined treatment with acetaminophen and Toximul MP8 had a statistically significant higher mortality rate than with the mice given influenza B alone. In weanling mice, it appeared that ASA shortened the time until death; however, this difference was not statistically significant. In vitro studies demonstrated that both ASA and acetaminophen decreased the interferon-induced antiviral responses of cultured mammalian cells. CONCLUSION: Antipyretics have the potential to exacerbate the consequences of a viral infection, although the specific effects are subtle and appear to be age-related.

Cytokine-mediated down-regulation of CYP1A1 in Hepa1 cells.

The activation of host defense mechanisms down-regulates microsomal cytochrome P450 in cell culture, humans, and animals. Investigation into various aspects of this effect using in vivo models has yet to define clearly the role that cytokines play in this phenomenon. The mechanism of down-regulation by immunostimulants, such as lipopolysaccharide (LPS), is explored with an in vitro model, utilizing a murine hepatoma (Hepa1) and a murine macrophage (IC-21) cell line. It is hypothesized that down-regulation of P450 activity by immunostimulants involves the activation of immune cells and the subsequent release of cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). The effects of immunostimulation on P450 activity are assessed by ethoxyresorufin O-dealkylase, an assay that measures CYP1A activity in Hepa1 cells. Initial studies demonstrated that LPS added directly to hepatoma cells had no effect on the levels of CYP1A1 activity. In contrast, a significant down-regulation in CYP1A1 activity occurred when hepatoma cells were incubated with monocyte conditioned medium obtained by incubating LPS with IC-21 cells. When pentoxifylline, a TNF-alpha synthesis inhibitor, was co-administered with LPS to macrophages, the down-regulation of CYP1A1 activity was prevented. The direct administration of murine recombinant TNF-alpha to hepatoma cells resulted in a down-regulation of CYP1A1 activity. These results implicated the release of TNF-alpha from macrophages as an important step in the down-regulation of CYP1A1 by LPS.

Cytochrome P4501A1 and cytochrome P4501A2 are downregulated at both transcriptional and post-transcriptional levels by conditions resulting in interferon-alpha/beta induction.

The interferon mediated downregulation of constitutive and inducible cytochrome P450 enzymes occurs through a pretranslational mechanism which depresses the mRNA encoding cytochrome P450s. We measured the transcription rates of CYP1A genes and the turnover of CYP1A mRNA in rats treated with the interferon-alpha/beta inducer polyinosinic acid-polycytidylic acid. The rate of transcription of CYP1A1 and CYP1A2 genes was significantly decreased in hepatic nuclei isolated from male rats treated with polyinosinic acid-polycytidylic acid (10 mg/kg). In addition the rate of degradation of hepatic CYP1A1 and CYP1A2 mRNA was examined following the inhibition of de novo transcription by actinomycin D (1 mg/kg). Messenger RNA levels were analysed by Northern and slot blotting with a 1.2 kb murine CYP1A1 cDNA probe. Interferon significantly augmented the rate of loss of CYP1A1 and CYP1A2 mRNAs suggesting that post-transcriptional degradation of mRNA contributes to the pre-translational events that cause cytochrome P450 downregulation. These results support the involvement of both transcriptional and post-transcriptional mechanisms in the loss of cytochrome P450s mediated by interferon inducers.

Amlodipine inhibits rat microsomal cytochrome P450-mediated drug biotransformation.

Calcium channel antagonists have been shown to inhibit cytochrome P-450-mediated metabolism both in vitro and in vivo. The purpose of the present study was to examine the effect of amlodipine on a suite of rat hepatic microsomal cytochrome P-450 activities to determine the potential for drug interactions. In this study, amlodipine (0.05 and 0.5 mM) decreased CYP1A-mediated ethoxyresorufin O-deethylase activity in microsomes prepared from noninduced (56 and 73% inhibition) and pyridine-induced (30 and 51% inhibition) rats. Amlodipine reduced pentoxyresorufin O-deethylase activity (a marker for CYP2B) to 15% of control in incubations utilizing microsomes from phenobarbital-treated rats, but had no effect on this enzyme reaction in noninduced microsomes. The para-nitrophenol hydroxylase, erythromycin N-demethylase, and lauric acid omega and omega-1 hydroxylase activities were significantly inhibited by 1 mM amlodipine in both noninduced and induced microsomes. These results suggest that amlodipine inhibits a number of different P450 forms and therefore has the potential to inhibit the metabolism of a large number of drugs.

Fluoroquinolone antibiotics inhibit cytochrome P450-mediated microsomal drug metabolism in rat and human.

The fluoroquinolone antibacterial agents have gained widespread use in the treatment of a broad range of bacterial infections. We recently described a possible interaction concerning the concomitant use of cyclosporine A and norfloxacin in pediatric renal transplant patients. We examined the effect of two common fluoroquinolone antibiotics on cytochrome P450-mediated drug biotransformations in human and rat liver microsomes. Rats were pretreated with inducers, which increased the levels of the P450 isozymes CYP3A2, CYP1A, CYP2E1, and CYP4A1. Ciprofloxacin and norfloxacin significantly depressed the N-demethylation of erythromycin by CYP3A4 in human microsomes and by CYP3A2 in rat microsomes. The inhibition was determined to be competitive in nature in rat microsomes, with ciprofloxacin and norfloxacin both exhibiting similar Ki values of 2.0 and 2.3 mM, respectively. Ciprofloxacin and norfloxacin also inhibited ethoxyresorufin-O-dealkylase (CYP1A). In contrast, ciprofloxacin and norfloxacin did not inhibit the metabolism of substrates that are specific for the P450 isozymes CYP2E1 and CYP4A1. Rats treated chronically with norfloxacin revealed no alterations in hepatic CYP3A2 protein levels or activity. These studies in hepatic microsomes demonstrate that fluoroquinolones can decrease CYP3A- and CYP1A-mediated biotransformation by competitive inhibition and that they have the potential to cause drug interactions with agents metabolized by these enzymes.

Differential effect of pentoxifylline on lipopolysaccharide-induced downregulation of cytochrome P450.

It is now established that inflammatory stimuli such as lipopolysaccharides (LPS) and polyinosinic acid:polycytidylic (polyIC) suppress hepatic expression of cytochrome P450 (P450) genes in rat liver. Previous studies have suggested that LPS- or polyIC-induced downregulation of P450 was due to endogenously released inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1, interleukin-6, and interferons (IFNs). To improve our understanding of the role of inflammatory cytokines in mediating P450 depression, we investigated the possibility of preventing P450 downregulation with pentoxifylline. Pentoxifylline has been shown to inhibit LPS-induced TNF-alpha production by suppression of TNF-alpha gene expression. The present study shows that in uninduced male rats pentoxifylline selectively prevents the downregulation of microsomal P4501A2 and P4502B caused by LPS. No protective effect of pentoxifylline on the downregulation of P4502E1 and P4503A1/2 was observed. PolyIC-induced downregulation of P4501A2, P4502B, P4502E1, and P4503A1/2 was not affected by pentoxifylline. These results suggest that the LPS-induced downregulation of P4501A2 and P4502B is mediated to a large extent by TNF-alpha. Other cytokines might be involved in the suppression of P4502E1 and P4503A1/2. The fact that polyIC-induced downregulation is not protected by pentoxifylline is further evidence that this agent acts via a selective induction of IFNs.

The role of nitric oxide generation in interferon-evoked cytochrome P450 down-regulation.

The down-regulation of cytochrome P450 during host defence involves one or more mediators of unknown source and identity. Owing to striking similarities in the features that are involved in the production of NO and the features involved in the down-regulation of cytochrome P450 we hypothesized that NO could be a mediator in the interferon-evoked loss of cytochrome P450. In these experiments the nitric oxide synthase inhibitor L-nitroarginine (5 mg/kg) failed to protect mice from the down-regulation of cytochrome P450, ethoxyresorufin dealkylase and para-nitrophenol hydroxylase in the liver following administration of the interferon inducer polyinosinic.polycytidylic acid. A higher dose of L-nitroarginine (30 mg/kg) given every 12 h for 3 days also did not protect against cytochrome P450 losses. In addition, no down-regulation of the enzyme was observed in animals treated with multiple doses of the NO-generating drugs glyceryl trinitrate (16 and 80 mg/kg given every 4 h for 16 h) and nitroprusside (20 and 100 mg/kg given every 4 h for 16 h). These results indicate that the down-regulation of cytochrome P450 by interferon or its inducers probably does not involve NO as a required intermediary.