Biological definition of multiple chemical sensitivity from redox state and cytokine profiling and not from polymorphisms of xenobiotic-metabolizing enzymes

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Abstract

Background

Multiple chemical sensitivity (MCS) is a poorly clinically and biologically defined environment-associated syndrome. Although dysfunctions of phase I/phase II metabolizing enzymes and redox imbalance have been hypothesized, corresponding genetic and metabolic parameters in MCS have not been systematically examined.

Objectives

We sought for genetic, immunological, and metabolic markers in MCS.

Methods

We genotyped patients with diagnosis of MCS, suspected MCS and Italian healthy controls for allelic variants of cytochrome P450 isoforms (CYP2C9, CYP2C19, CYP2D6, and CYP3A5), UDP-glucuronosyl transferase (UGT1A1), and glutathione S-transferases (GSTP1, GSTM1, and GSTT1). Erythrocyte membrane fatty acids, antioxidant (catalase, superoxide dismutase (SOD)) and glutathione metabolizing (GST, glutathione peroxidase (Gpx)) enzymes, whole blood chemiluminescence, total antioxidant capacity, levels of nitrites/nitrates, glutathione, HNE-protein adducts, and a wide spectrum of cytokines in the plasma were determined.

Results

Allele and genotype frequencies of CYPs, UGT, GSTM, GSTT, and GSTP were similar in the Italian MCS patients and in the control populations. The activities of erythrocyte catalase and GST were lower, whereas Gpx was higher than normal. Both reduced and oxidised glutathione were decreased, whereas nitrites/nitrates were increased in the MCS groups. The MCS fatty acid profile was shifted to saturated compartment and IFNgamma, IL-8, IL-10, MCP-1, PDGFbb, and VEGF were increased.

Conclusions

Altered redox and cytokine patterns suggest inhibition of expression/activity of metabolizing and antioxidant enzymes in MCS. Metabolic parameters indicating accelerated lipid oxidation, increased nitric oxide production and glutathione depletion in combination with increased plasma inflammatory cytokines should be considered in biological definition and diagnosis of MCS.

Introduction

The question whether multiple chemical sensitivity (MCS) and other medically unexplained syndromes such as fibromyagia, chronic fatigue, Persian Gulf War, sick building syndrome, etc.now also collectively labelled “idiopathic environmental intolerances”are clinically defined entities has remained unanswered so far. This is due to the broad symptom constellation, the lack of clear-cut diagnostic criteria and dose-dependency of clinical symptoms upon exposure, and the poor knowledge about pathogenic mechanisms (Bolt and Kiesswetter, 2002, Bornschein et al., 2001, Kipen and Fiedler, 2002). As the number of subjects affected by MCS in developed countries has been growing steadily, reaching up to 15% of the population in the United States (Caress and Steinemann, 2003), also the public concern has increased, as exemplified by new alarming definitions such as “chemical AIDS,” “chemical allergy,” “twenties century disease,” etc. Patients self-reporting MCS suffer recurring multi-organ symptoms affecting nervous, cardio-vascular, gastro-intestinal, respiratory, and skeletal-muscular systems, skin and ocular epithelia (Kipen and Fiedler, 2002). In accord with epidemiological surveys performed in the United States and Canada, the symptoms of pathologically increased chemical sensitivity are fairly prevalent in the general population, being more frequent and severe in the subjects with bronchial asthma (Kipen and Fiedler, 2002, Kreutzer et al., 1999). Majority of MCS patients develop first symptoms after heavy exposure to odorous organic substances (primary triggering event). The recurrence of the same symptoms and/or appearance of new ones occurs following exposures to extremely low, sometimes negligible, concentrations of everyday odours such as perfume, fresh paint, cleaning chemicals, newsprint, new carpeting, and numerous other organic chemistry products (low level triggers) (Caress and Steinemann, 2003, Mattson and Cheng, 2006 Willette et al., 2007, Yadav et al., 2007). Some publications ascribed the MCS syndrome to somatoform disorders or other psychiatric disturbances (Bailer et al., 2005). Others attempted to prove that extreme individual sensitivity in MCS could be due to the inherited impairment in xenobiotics/endobiotics metabolism through phase I (McKeown-Eyssen et al., 2004) and phase II enzymes (Schnakenberg et al., 2007), although this hypothesis is far to be conclusively demonstrated (Berg et al., 2010). A vicious cycle has been hypothesized (Pall, 2001), including peroxynitrite overproduction, lipid peroxidation, and the induction of a pro-inflammatory condition, this latter leading in turn to an increased nitric oxide (NO) plus superoxide generation, followed by additional peroxynitrite formation. No other data on genetic, immune, or metabolic disturbances in MCS patients have been retrieved from available databases.

Here, we sought to prove our working hypothesis on the inherited or/and acquired dysfunction of the chemical defensive system as a molecular basis for MCS (Korkina et al., 2009). The ubiquitous chemical defense network (phase I–II xenobiotic-metabolizing and antioxidant enzymes) appears very early in the evolution and handles low molecular weight inorganic and organic xenobiotics, as well as endogenous non-protein signaling molecules, mediators of inflammation, degradation products, and toxic products of cellular metabolism (Goldstone et al., 2006). Taking into consideration the reciprocal regulation of the chemical defensive and immune systems, exerted mainly through cytokine-mediated interactions (Crawford et al., 1997, Haarmann-Stemmann et al., 2009, Kerkvliet, 2009), we focused on both possible genetic and metabolic markers of chemical defensive system dysfunctions and cytokine dysregulation in MCS patients. To evaluate this hypothesis, we compared genetic polymorphisms of genes encoding drug/xenobiotic metabolizing enzymes (cytochrome P450 isoforms (CYPs), UDP-glucuronosyl transferase (UDT), and glutathione S-transferases (GSTs)), and parameters related to redox status, fatty acid and cytokine patterns, in the blood components of Italian MCS patients, both diagnosed and suspected, and of healthy Italian age and sex matched subjects. On the grounds of the results obtained, we suggest that there exist serious and multiple dysfunctions of chemical defensive system in MCS patients. These dysfunctions may mainly depend not on genetic defects but on non-genetic modifications of metabolizing/antioxidant enzyme expression and/or activity, mediated by redox active agents such as NO and inflammatory cytokines, for example IFNgamma and IL-10.

Section snippets

Reagents and antibodies

Majority of chemical reagents, HPLC standards, mediums, reverse transcription polymerase chain reaction (RT PCR) primers for GSTs, and fluorogenic probes were from Sigma Chemical Co. (St. Louis, MO, USA); Gpx and NO2/NO3 kits from Cayman Chem. Co. (Ann Arbor, MI, USA); fluorescent probe-labelled anti-cytokine antibodies were from Bio-Rad Laboratories Inc. (Hercules, CA, USA); DNA extraction kit was from Qiagen (Hilden, Germany), and PCR kit for CYPs and UGT from Applied Biosystems Inc.

Allele and genotype frequencies of CYPs and UGT

Allele and genotype frequencies of CYPs (CYP2C9, CYP2C19, CYP2D6, and CYP3A5) and UGT among a subgroup of MCS patients (n = 110) and healthy controls (n = 218) are shown in Table 2, Table 3. Genotype frequencies among both cases and controls were in equilibrium with the Hardy-Weinberg equation, and similarly, allele frequencies among cases and controls were within the 95% confidence intervals based on healthy control frequencies. No statistically significant differences were observed between the

Discussion

The polymorphism in genes encoding both metabolizing enzymes and the receptors and transcriptional factors regulating their expression, may account for the existing inter-individual variations in xenobiotics metabolizing activity, and it has been suggested as a possible mechanism underlying MCS. On the other hand, individual peculiarity of adaptive response to chemical stressors at the epigenetic level through the direct interaction of these substances and their metabolites with biologically

Acknowledgments

This work was supported by grants from Italian Ministry for Health (IDI IRCCS-RC2008), Italian Ministry for University and Research (MIUR-UNISI-2008), the Swedish Medical Society and the Swedish Research Council. Excellent technical assistance of Mr. Luciano Innocenzi is gratefully appreciated.

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