Overview on legislation and scientific approaches for risk assessment of combined exposure to multiple chemicals: the potential EuroMix contribution.

This article reviews the current legislative requirements for risk assessment of combined exposure to multiple chemicals via multiple exposure routes, focusing on human health and particularly on food-related chemicals. The aim is to identify regulatory needs and current approaches for this type of risk assessment as well as challenges of the implementation of appropriate and harmonized guidance at international level. It provides an overview of the current legal requirements in the European Union (EU), the United States and Canada. Substantial differences were identified in the legal requirements for risk assessment of combined exposure to multiple chemicals and its implementation between EU and non-EU countries and across several regulatory sectors. Frameworks currently proposed and in use for assessing risks from combined exposure to multiple chemicals via multiple routes and different durations of exposure are summarized. In order to avoid significant discrepancies between regulatory sectors or countries, the approach for assessing risks of combined exposure should be based on similar principles for all types of chemicals. OECD and EFSA identified the development of harmonized methodologies for combined exposure to multiple chemicals as a key priority area. The Horizon 2020 project "EuroMix" aims to contribute to the further development of internationally harmonized approaches for such risk assessments by the development of an integrated test strategy using in vitro and in silico tests verified for chemical mixtures based on more appropriate data on potential combined effects. These approaches and testing strategies should be integrated in a scientifically based weight of evidence approach to account for complexity and uncertainty, to improve risk assessment.

Interleukin-6 mediated upregulation of CYP1B1 and CYP2E1 in colorectal cancer involves DNA methylation, miR27b and STAT3.

BACKGROUND: The pro-inflammatory cytokine interleukin-6 (IL6) promotes colorectal cancer (CRC) development. It is also known to regulate cytochrome P450 (CYP450) enzymes, which are involved in CRC tumour initiation and promotion via activation of chemical carcinogens. Here, IL6 regulation of CYP450 expression was investigated in CRC. METHODS: The effect of IL6 on CYP 1A1, 1B1 and 2E1 expression was determined in vitro using CRC cell lines HCT116 and SW480, and CYP450 expression was determined by immunohistochemistry in CRC tissues previously shown to have increased levels of IL6. RESULTS: In mechanistic studies, IL6 treatment significantly induced CYP1B1 and CYP2E1, but not CYP1A1, gene expression in HCT116 and SW480 cells. CYP2E1 expression regulation occurred via a transcriptional mechanism involving STAT3. For CYP1B1 regulation, IL6 downregulated the CYP1B1-targeting microRNA miR27b through a mechanism involving DNA methylation. In clinical samples, the expression of CYP1B1 and CYP2E1, but not CYP1A1, was significantly increased in malignant tissue overexpressing IL6 compared with matched adjacent normal tissue. CONCLUSIONS: Colonic inflammation with the presence of IL6 associated with neoplastic tissue can alter metabolic competency of epithelial cells by manipulating CYP2E1 and CYP1B1 expression through transcriptional and epigenetic mechanisms. This can lead to increased activation of dietary carcinogens and DNA damage, thus promoting colorectal carcinogenesis.

Searching for novel biomarkers of centrally and peripehrally-acting neurotoxicants, using surface-enhanced laser desorption/ionisation-time-of-flight mass spectrometry (SELDI-TOF MS).

The neurotoxicity of chemicals to humans is difficult to monitor as there are no suitable methods of detecting early neuronal dysfunction. Here, a proof of principle study was designed to assess the potential of identifying protein biomarkers in accessible biofluids for this purpose. Groups of rats were treated with a range of doses of the model neurotoxicants, acrylamide (0, 2, 10, 50mg/kg) and methylmercury (0, 0.2, 1, 5mg/kg) for up to 3 weeks and samples of serum, urine, and cerebral spinal fluid analysed by surface-enhanced laser desorption/ionisation-time-of-flight mass spectrometry. There was no neuropathology up to the highest dose tested. Protein profiles were obtained from all samples and changes in the levels of many proteins were detected in both serum and urine, although not cerebral spinal fluid. In serum, the combination of three protein ion levels with m/z values of 4968, 9402 and 12,948 was able to correctly classify the treatment groups thus: 88% control, 100% acrylamide, 92% methylmercury. In urine, three protein ions with m/z values of 4944, 12,966 and 21,992 classified correctly the groups: 67% control, 94% acrylamide, 97% methylmercury. Similar classifications using other serum and urinary protein ions were also possible. This indicates the potential of serum and urine protein biomarkers for the assessment of sub-clinical neurotoxicity.

The food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine activates S-phase checkpoint and apoptosis, and induces gene mutation in human lymphoblastoid TK6 cells.

The mutagenic heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-blpyridine (PhIP) is formed at parts per billion levels when meat is cooked. It is efficiently absorbed from cooked food and extensively activated to its genotoxic N-hydroxy derivative by human cytochrome P4501A enzymes. It is also a rodent carcinogen. To better understand the genetic toxicity of PhIP, we have examined its effect on the cell cycle and gene mutation frequency using human lymphoblastoid cells (TK6) as a model. Because TK6 cells are unable to activate PhIP, we have cultured the cells in the presence of irradiated Chinese hamster XEMh1A2-MZ cells that have been genetically engineered to express human CYP1A2. Asynchronized TK6 cells were harvested at various times after treatment with PhIP (1.25-10 microg/ml), fixed and stained with propidium iodide for the examination of cell cycle by fluorescence-activated flow cytometry. After 20 h of PhIP treatment, a slight S-phase delay of the cell cycle was observed. Normal cell cycle recovered after the cells were washed and further cultured in the absence of PhIP for 5 days. However, PhIP treatment for 40 h induced a more pronounced S-phase arrest that was accompanied by a decrease in the level of cyclin A, an S-phase cyclin. This was followed by the appearance of a sub-G1 population (indicative of apoptotic cell death), range from 13 to 54% with PhIP concentrations from 1.25 to 10 microg/ml, compared with 5% in the vehicle control. A concomitant increase of mutation frequency at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, assessed by colony formation assay in the presence of 6-thioguanine, was detected after 40 h-range, 16 to 45 x 10(-6) compared with 12 x 10(-6) in cultures without PhIP. In G1-enriched cell populations (synchronized culture), although PhIP induced S-phase delay, the induction of sub-G1 cells was substantially decreased. Our studies show that in TK6 cells, PhIP activates S-phase checkpoint, yet eludes G1 and G2-M checkpoints, and is accompanied by increased apoptosis and gene mutation. If treatment with PhIP induces similar cellular reactions in vivo, then activation of S-phase checkpoint with avoidance of G1 and G2-M checkpoints could be important factors in PhIP-induced genetic damage and neoplastic disease.

Intra- and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef.

During the cooking of beef, the genotoxic heterocyclic aromatic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed. Little is known about the fate of these compounds in humans or the factors affecting it. We have developed assays based on capillary column gas chromatography-negative ion mass spectrometry capable of the simultaneous measurement of MeIQx, DiMeIQx, and PhIP in cooked meat and in human urine using stable isotope labeled analogues. Ten normal, healthy male volunteers were invited to consume a standard cooked meat meal (400-450 g lean beef, cooked as patties on a griddle hotplate) on four separate occasions over a period of 14 months. Following consumption of the test meals, urine was collected from 0 to 8 h, during which time all free amines were excreted and analyzed for MeIQx, DiMeIQx, and PhIP. Subjects ingested 240 +/- 9 (SEM) g cooked meat, which contained 2.2 +/- 0.2 ng MeIQx/g meat, 0.7 +/- 0.1 ng DiMeIQx/g meat, and 16.4 +/- 2.1 ng PhIP/g meat. The variability in relative systemic bioavailability was assessed from the percentage of ingested amine excreted unchanged in the urine. Subjects excreted 2.1 +/- 1.1% of MeIQx and 1.1 +/- 0.5% of PhIP ingested as unchanged amine in the urine. Levels of DiMeIQx in urine, if present, were below the sensitivity of our assay (20 pg/ml) and could not be detected in any of the samples analyzed. Irrespective of dose, urinary excretion of unchanged MeIQx or PhIP (expressed as a percentage of the ingested dose) remained constant for each individual subject. The intraindividual coefficients of variation for MeIQx (28.4%) and PhIP (23.7%) were low and the pooled interday (intrasubject) coefficients of variation for both compounds were only 19 and 3.4%, respectively. In contrast, inter-subject (intraday) variation was greater, with pooled coefficients of variation of 145% for MeIQx and 71% for PhIP. Based on these studies, it should be possible to use the percentage excretion of MeIQx and PhIP to assess the relative bioavailability of these compounds in humans.

Mutagenesis of mouse intestine in vivo using the Dlb-1 specific locus test: studies with 1,2-dimethylhydrazine, dimethylnitrosamine, and the dietary mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline.

The ability of three model carcinogens, 1,2-dimethylhydrazine, dimethylnitrosamine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, to induce mutation in a novel in vivo assay in mouse intestine has been examined. The assay is based on mutations at the Dlb-1 locus which determines the tissue specific pattern of expressio of the binding site for the lectin Dolichos biflorus agglutinin. In C57BL/6J x SWR F1 mice Dlb-1 mutants are recognized as clones of epithelial cells not staining with a peroxidase conjugate of D. biflorus agglutinin. Chronic administration of 1,2-dimethylhydrazine (20 mg/kg/week s.c. for 10 weeks) induced Dlb-1 mutants, whereas administration of a single dose did not. Similarly, chronic dimethylnitrosamine treatment p.o. (0.001% in drinking water for 8 weeks) induced Dlb-1 mutants, but acute administration did not. In contrast, neither chronic nor acute treatment of the mice with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline induced Dlb-1 mutations. The activities of 1,2-dimethylhydrazine, dimethylnitrosamine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the Dlb-1 assay more accurately reflect their carcinogenic potential than do many in vitro bioassays.

Relationship between oxidative metabolism of 2-acetylaminofluorene, debrisoquine, bufuralol, and aldrin in human liver microsomes.

The capacity of human liver microsomes from 28 individuals to metabolize debrisoquine and bufuralol, two drugs oxidized polymorphically in humans, as well as the carcinogen 2-acetylaminofluorene (AAF), was determined. In addition, the cytochrome P-450 content and the capacity of these microsomes to carry out the epoxidation of aldrin were measured. Interindividual differences in debrisoquine 4-hydroxylation, bufuralol 1-hydroxylation, and aldrin epoxidation were 12-, 20-, and 2.4-fold, respectively. The metabolism of debrisoquine was not correlated with cytochrome P-450 content (r = 0.26), whereas both the metabolism of bufuralol (r = 0.45; r2 = 0.20) and the epoxidation of aldrin (r = 0.72; r2 = 0.52) were correlated. Rates of debrisoquine and bufuralol metabolism were significantly correlated (r = 0.73), whereas only weak correlations existed between debrisoquine:aldrin (r = 0.49) and bufuralol:aldrin (r = 0.51). Because biphasic kinetics have been observed in human liver microsomes for the 7- and 5-hydroxylation of AAF, two concentrations of this substrate were used. The disappearance of AAF at either 0.37 or 50 microM was not correlated with debrisoquine, bufuralol, or aldrin metabolism. Similarly, at 0.37 microM AAF, no correlation existed between the formation of N-, 1-, 3-, 5-, 7-, and 9-hydroxylation products of AAF and debrisoquine, bufuralol, or aldrin metabolism. At 50 microM AAF, only the 7-hydroxylation of this substrate correlated with bufuralol metabolism (r = 0.47). This lack of, or weak correlation between pathways leading to metabolic activation (N-hydroxylation) or detoxication (C-hydroxylation) of the carcinogen AAF and debrisoquine, bufuralol, and aldrin metabolism strongly suggests that different forms of cytochrome P-450 are involved in these pathways. In contrast, exceptionally high correlations (r greater than 0.94) existed between N-OH-AAF:1-OH-AAF. N-OH-AAF:7-OH-AAF, and 7-OH-AAF:1-OH-AAF at the low concentration of AAF, and imply that similar forms of cytochrome P-450 produce these metabolites. However, at 50 microM AAF, these correlations are considerably weaker and explain less than 35% of the variance in the data. It is concluded, based on these multiple cross-correlations, that common cytochrome P-450 isoenzymes are involved in the formation of AAF metabolites, while the metabolism of debrisoquine, bufuralol, and aldrin is unrelated to the metabolism of this carcinogen in human liver microsomes.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a potent mutagen in the mouse small intestine.

Mutations in long lived stem cells are critical events in carcinogenesis. The Dlb-1 assay detects intestinal stem cell mutation at the Dlb-1 locus in Dlb-1a/b heterozygous mice by visualizing mutated clones of epithelial cells in situ which do not bind the lectin Dolichos biflorus agglutinin. We have used this assay to show that the food-derived heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent intestinal mutagen when administered either i.p. or p.o. This contrasts with the inactivity of the structurally related mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the assay which we have described previously. Immunocytochemical localization of the P-450 enzyme CYP1A2, which is responsible for the primary activation of these mutagens, shows that in untreated mice it is present in liver hepatocytes and in occasional villus epithelial cells but is absent from the target intestinal stem cell population. In addition, liver microsomes, unlike intestinal microsomes, were able to convert PhIP to the proximate mutagen N-hydroxy-PhIP. CYP1A2 immunoreactivity in beta-napthoflavone-induced animals was elevated in liver hepatocytes and increased to a lesser extent in duodenal villus epithelial cells. Treatment with beta-napthoflavone produced an unexpected 46% decrease in the number of Dlb-1 mutations in response to PhIP. Following treatment with PhIP, there was no difference in the number of Dlb-1 locus mutations between the proximal and distal ends of the small intestine in uninduced animals, indicating that the bile duct is unlikely to be responsible for transport of mutation inducing metabolites of PhIP to the small intestine. Our results demonstrate that metabolic activation of an indirect acting genotoxic agent can occur at a site other than the target tissue, and absence of the enzymes required for activation of a mutagen does not necessarily protect that tissue from its genotoxic effects.

CYP1A2-catalyzed conversion of dietary heterocyclic amines to their proximate carcinogens is their major route of metabolism in humans.

The contribution of CYP1A2 to the metabolism of the dietary heterocyclic amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in vivo in humans, has been determined with furafylline, a highly selective inhibitor of this enzyme. The inhibitory potential of furafylline in vivo was first assessed by determining its effect on clearance of phenacetin to paracetamol by the model CYP1A2-dependent O-deethylation pathway. Furafylline inhibited this reaction by > 99% in all subjects, thus demonstrating its applicability to determining the contribution of CYP1A2 to a given reaction in vivo. A group of 6 healthy male volunteers received either placebo or 125 mg furafylline, in a double-blind balanced crossover design, 2 h prior to consuming a test meal of fried beef containing a known amount of amines. The excretion of PhIP and MeIQx in urine was determined during the subsequent 28 h, using gas chromatography-mass spectrometry. Following furafylline, the excretion of unchanged MeIQx increased 14.3-fold, while that of PhIP increased 4.1-fold (P < 0.01, paired t test). Elimination of both amines was first order and very rapid, with half-lives of < 5 h. The elimination rate constants did not change following furafylline, suggesting that total clearance is limited by hepatic blood flow. Because the elimination of the amines was first order, it was possible to calculate the contribution of CYP1A2 to the clearance of the amines. CYP1A2-catalyzed metabolism accounts for 91% of the elimination of ingested MeIQx and 70% of ingested PhIP, most likely via N-hydroxylation.

An inhibitory monoclonal anti-protein antibody and an anti-peptide antibody share an epitope on rat cytochrome P-450 enzymes CYP1A1 and CYP1A2.

A monoclonal antibody, 12/2/3/2, which was raised against purified rat CYP1A1 recognises specifically rat and mouse CYP1A1 and CYP1A2, but not any cytochrome P-450 present in hepatic microsomal fractions from rabbit, guinea pig, hamster or human. By comparing the primary sequences of cytochromes P-450 to which 12/2/3/2 does and does not bind, 10 possible locations for its epitope were found. Of these, one was extremely hydrophilic and, hence, predicted to be the most antigenic in the native protein. An antibody was produced against the synthetic peptide corresponding to this region (Gly-Arg-Asp-Arg-Gln-Pro-Arg-Leu: residues 356-363 and 350-357 of rat CYP1A1 and CYP1A2, respectively). The antibody bound to rat, mouse and hamster CYP1A1 and to rat and mouse CYP1A2, but did not bind to any protein present in hepatic microsomal fractions from the rabbit, guinea pig or human. The binding of the anti-peptide antibody to CYP1A1 or CYP1A2 was partially antagonised by the monoclonal antibody. However, whereas the monoclonal antibody inhibited both CYP1A1- (aryl hydrocarbon hydroxylase) and CYP1A2-(high-affinity phenacetin O-deethylase) dependent monooxygenase activity, the anti-peptide antibody was without effect on these activities. Antigen denaturation by 8 M urea or 0.05% (w/v) SDS had no effect on binding of the anti-peptide antibody to cytochrome P-450, whilst binding of the monoclonal antibody was reduced by more than 1000-fold. The anti-peptide antibody partially antagonised the binding of 12/2/3/2 to urea-denatured but not native cytochrome P-450. These data suggest that whilst the complete binding site for the monoclonal antibody is discontinuous, sufficient of the epitope is linear, so that when the antigen is denatured the monoclonal antibody is still able to bind and this binding is antagonised by the anti-peptide antibody. However, inhibition of catalytic activity by the monoclonal antibody must require binding to discontinuous residues.

Assessment of P450 induction in the marmoset monkey using targeted anti-peptide antibodies.

The identity and expression of hepatic P450 enzymes in marmosets was investigated using a panel of anti-peptide antibodies originally targeted against human P450 enzymes. In immunoblotting, of 12 antibodies examined, 10 bound specifically to bands in marmoset liver microsomal fraction corresponding to P450 enzymes. It is proposed that these represent marmoset CYP1A1, CYP1A2, CYP2A, CYP2B, CYP2C forms (CYP2C-1 and CYP2C-2), CYP2D19, CYP3A21 and another CYP3A form (CYP3A-m). The antibodies, together with an anti-marmoset CYP2E1 antibody, were used to investigate the expression of 10 P450 enzymes in marmosets treated with P450-inducing chemicals. Treatment with phenobarbitone caused CYP2B, CYP2C-2 and CYP3A21 levels to increase, rifampicin caused increases in CYP2B and CYP2C-1 and a decrease in CYP3A21 levels, whereas dioxin caused CYP1A1 and CYP1A2 levels to increase and CYP2E1 levels to decrease. Clofibric acid did not induce any P450. P450 enzyme activities were assessed using 8 different substrates and increases were found after treatment with phenobarbitone, rifampicin, and dioxin. However, due to species differences in substrate selectivity, it proved difficult to ascribe these changes to individual P450 enzymes. Thus, the use of anti-peptide antibodies provides a more informative way of assessing the levels of specific P450 enzymes than enzyme activity measurements.

Mechanisms of cell death.

There is increasing evidence that the mechanisms of chemically mediated cell death are common to a wide variety of cell types and to a large number of toxic compounds. The perturbation of Ca2+ homeostasis appears to be particularly important and may be due to modification of SH-groups in key enzymes. Donald Davies and colleagues discuss the mechanisms by which early events induced by exposure to toxic chemicals may lead to these changes, and their possible consequences. It is now clear that reduced glutathione plays a pivotal role, not only in detoxifying reactive compounds but also in reversing the early biochemical changes in the cell.

Polymorphic debrisoquine 4-hydroxylase activity in the rat is due to differences in CYP2D2 expression.

The female Dark Agouti rat is widely used as an animal model for the CYP2D6 poor metabolizer phenotype, males of other strains such as Sprague Dawley or Wistar serving as models for the extensive metabolizer phenotype. To determine the relative level of expression of CYP2D enzymes in the liver of female and male Dark Agouti, Sprague Dawley and Wistar rats, anti-peptide antibodies were raised in rabbits against short synthetic peptides representing the C-termini of the rat P450 enzymes CYP2D1, CYP2D2, CYP2D3, CYP2D4 and CYP2D5. In immunoblotting studies, it was found that the hepatic expression of CYP2D1 was greater in Dark Agouti rats than Sprague Dawley or Wistar rats. In contrast, hepatic CYP2D2 was 30-40-fold less abundant in female Dark Agouti than female Sprague Dawley or Wistar rats and six- to eightfold less abundant in male Dark Agouti than male Sprague Dawley or Wistar rats. No hepatic CYP2D3 could be detected in either sex of any of the three strains. Hepatic CYP2D4 expression was generally greater in male than female rats, and higher in Dark Agouti compared with Sprague Dawley or Wistar strains. CYP2D5 was expressed in the livers of female and male Dark Agouti rats but not in female Sprague Dawley or Wistar rats. This form was variably expressed in livers of male Sprague Dawley and Wistar rats. Hepatic debrisoquine 4-hydroxylase activity was markedly reduced in female and male Dark Agouti rats as compared to Sprague Dawley or Wistar rats and correlated (r = 0.88; P < 0.001) with the hepatic CYP2D2 content. Recombinant CYP2D2 was 18-fold more active at catalysing the 4-hydroxylation of debrisoquine than CYP2D1. Furthermore, quinine markedly inhibited CYP2D2-mediated debrisoquine and metoprolol oxidation, while quinidine, its diastereoisomer, inhibited the reactions to a lesser extent. In conclusion, these results show that impaired debrisoquine 4-hydroxylase activity in the female Dark Agouti rat is due to low levels of CYP2D2.

Effect of isoniazid on vitamin D metabolism and hepatic monooxygenase activity.

isoniazid, 300 mg daily for 14 days, reduced serum calcium and phosphate levels (P less than 0.001) in eight healthy subjects. After a single dose of isoniazid the concentration of 1 alpha-,25-dihydroxyvitamin D, the most active metabolite of vitamin D, fell by 47% (P less than 0.01) and was reduced throughout the study. Levels of 25-hydroxyvitamin D, the major circulating form of the vitamin, declined in all subjects and to below normal range in six (P less than 0.01). Parathyroid hormone levels rose by 36% (P less than 0.01) in response to the relative hypocalcemia produced. Isoniazid inhibited hepatic mixed-function oxidase activity, as evidenced by a reduction in antipyrine and cortisol oxidation, and a similar inhibition of the hepatic 25-hydroxylase and renal 1 alpha-hydroxylase would explain the reduction in the corresponding vitamin D metabolites. This perturbation of vitamin D metabolism differs from the vitamin D wasting effects after rifampicin. Patients with tuberculosis treated with isoniazid and rifampicin may show changes similar to those shown here in calcium and phosphate homeostasis and thus may be at risk of developing metabolic bone disorders.

Effect of rifampicin and isoniazid on vitamin D metabolism.

Rifampicin, 600 mg, and isoniazid, 300 mg daily for 14 days, reduced circulating levels of 25-hydroxy vitamin D (25-OHD) and 1 alpha, 25-dihydroxy vitamin D (1,25(OH)2D) by 34% (P less than 0.01) and 23% (P less than 0.05) in eight healthy subjects. This was accompanied by a rise in parathyroid hormone (PTH) of 57% (P less than 0.01), but not by a fall in serum calcium or phosphate levels. There was induction of endogenous cortisol oxidation in all subjects, but only in four fast acetylators was there a concomitant increase in antipyrine elimination. In the four slow acetylators antipyrine metabolism was inhibited after the first dose of the drugs. In nine tuberculous patients followed serially there was a fall in 25-OHD and 1,25 (OD)2D and a rise in PTH at the end of 1 mo (P less than 0.05). After 6 mo therapy 25-OHD concentration was further reduced (P less than 0.01), but there was no significant change in 1,25 (OH)2D or PTH levels. Combination treatment with rifampicin and isoniazid perturbs vitamin D metabolism, but less than might have been predicted from reports on each drug given alone. Nevertheless, tuberculous patients with already compromised calcium homeostasis receiving this combination of drugs should be carefully monitored.

Rifampicin and vitamin D metabolism.

A 2-wk course of rifampicin orally (600 mg/day) in 8 male subjects resulted in a consistent fall in plasma 25-hydroxycholecalciferol (25-OHD) levels of around 70%, accompanied by increased oxidation of antipyrine and 6 beta-hydroxycortisol (indicative of hepatic enzyme induction). Plasma levels of 1,25-dihydroxycholecalciferol, parathyroid hormone, and calcitonin were not altered. The fall in 25-OHD may represent the earliest lesion of drug-induced osteomalacia.

Cross-reaction of antibodies to coupling groups used in the production of anti-peptide antibodies.

A polyclonal antibody raised against a peptide conjugated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride to maleic anhydride-derivatised lysozyme showed substantial cross-reactivity with m-maleimidobenzoyl-N-hydroxysuccinimide ester-derivatised haemocyanin. This was due to antibodies produced against maleic anhydride-derivatised groups on lysozyme that reacted with m-maleimidobenzoyl-N-hydroxysuccinimide ester-derivatised groups on haemocyanin. This observation is important because it is common practice, in the production of anti-peptide antibodies, to use two conjugates. The same peptide is coupled to two different protein carriers by two different coupling methods. One conjugate is used for immunisation and the other for testing the serum. This method assumes that the only antigen common to the two conjugates is the peptide and this was not the case here. A method is described for screening sera which involves affinity purification of the anti-peptide antibody and comparison of binding to the immunogen with that to an appropriate control conjugate. This method avoids the problem of any cross-reaction to coupling groups or proteins.

Co-localization of P450 enzymes in the rat substantia nigra with tyrosine hydroxylase.

Susceptibility to develop Parkinson's disease has been linked to abnormalities of P450 enzyme function. Multiple P450 enzymes are expressed in brain but the relationship of these to Parkinson's disease is unknown. We have investigated the expression of P450 enzymes in the rat substantia nigra and their co-localization in tyrosine hydroxylase-positive neurons and astrocytes. Immunohistochemistry was performed using anti-peptide antisera against the following P450 enzymes: CYP1A1, CYP1A2, CYP2B1/2, CYP2C12, CYP2C13/2C6, CYP2D1, CYP2D4, CYP2E1, CYP3A1, CYP3A2 and NADPH-P450 oxidoreductase. Immunoreactivity in nigral cells was found only for CYP2E1 and CYP2C13/2C6. CYP2E1 immunoreactivity was localized to many midbrain nuclei including the substantia nigra pars compacta but not the substantia nigra pars reticulata while immunoreactivity to CYP2C13/2C6 was found in the substantia nigra pars compacta, substantia nigra pars reticulata and many other midbrain nuclei. Sections of rat midbrain double labelled for either CYP2E1 or CYP2C13/2C6 and tyrosine hydroxylase or glial fibrillary acidic protein were examined for co-localization by confocal laser scanning microscopy. CYP2E1 and CYP2C13/2C6 immunoreactivity was found in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta but not in glial cells. CYP2C13/2C6, but not CYP2E1, was also found in non-glial, non-tyrosine hydroxylase-expressing cells in the substantia nigra pars reticulata. Isoniazid induction increased CYP2E1 fluorescence signal intensity from nigral dopaminergic neurons. At least two P450 enzymes are found in nigral dopamine containing cells and one, namely CYP2E1, is selectively localized to this cell population. CYP2E1 is a potent generator of free radicals which may contribute to nigral pathology in Parkinson's disease. The expression of CYP2E1 in dopaminergic neurons in substantia nigra raises the possibility of a causal association with Parkinson's disease.