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Multiple chemical sensitivity seen from physiological and genetic properties of human populations affected by chemical stress

Karl-Rainer Fabig*

* Practicioner (Hamburg). - The presentation was given  at  the second day of the “Workshop of the Thematic Network SUSTAINABILITY STRATEGY” ( www.sustainability-strategy.net   )  „From sustainability science to sustainability governance Proposals for an improvement of European sustainability strategy elaboration and implementation” which was held from  01.-03.12.2004 in Roskilde and Copenhavn

Abstract

Introduction

Chemical-induced diseases  are medical conditions of concern. The reasons why some patients develop clinical symptoms due to low-dose background exposure  are completely unknown. This leads to an increased error ratio of individuals with „chemical-related symptoms” (CRS) to be patients of a psychiatric or any other disease.

Methods

1.143 (41.6 percent) of  all 2.746 patients (visitors of my practise in Hamburg) between January 2000 and December 2003 answered a validated questionnaire about chemical symptoms called “modified QEESI”. The primary aim was the measurement and documentation of the scores of the first ten items named “sensitizing capabilities of chemicals” (SCC). Then - without any  medical information - the  molecularbiologist Dr. Eckart Schnakenberg (university of Bremen, director of the “Institute for Pharmacogenetics and Genetic Disposition” - IPGD) analyzed  the gene variants of the enzymes N-acetyltransferase 2, glutathione S-transferase M1, glutathione S-transferase T1.  The single nucleotide polymorphisms of  these genes of phase II in xenobiotic metabolism was analysed for  861 patients. After exclusion because age <20 years or age >90 years, psychiatrical and/or neurological diseases and ethnical causes  remained 800 caucasian  individuals for the case-control-study.

Results 

The single nucleotide polymorphisms of these genes and the eight possible gene variants combinations - because of dichotomy of the phenotypes of enzymes - correlated significantly with the reported sensitizing capability of chemicals ( F=30.52; p < 0.000). The modification of  the SCC through gene variants was seen in people with no chemical exposure (exposure in background). After chemical exposure - measured by biomonitoring and/or ambiente monitoring - the SCC effects were stronger.

Conclusion

The observations give evidence that single nucleotide polymorphisms within these genes contribute to an individual risk for the development of chemical-related symptoms. However, these results can help to identify genetic influences in patients suffering from chemical-related symptoms and reduce the number of misclassified patients. In a political way the findings may modify the sustainability-strategy and plans of the evaluation of  chemicals in the REACH process of the European Union.

Key words: questionnaire, sensitizing capability of chemicals, chemical-related symptoms,  multiple chemical sensitivity syndrome, background exposure, susceptibility, single nucleotide polymorphism,  gene variants

Abbreviations:

Introduction

Chemical-induced diseases are a clinical entity of unknown origin. For more than hundred years it has been observed that chemicals like drugs and occupationally used substances may induce severe side reactions in human beings. Rehn was the first scientist who described in 1895 the importance of occupationally used chemicals as aetiological factors involved in the development of urogenital tract tumors (Rehn 1895). He identified the frequently used chemical substance aniline for releasing bladder cancer. Later the contamination of aniline by 2-naphthylamine was identified as risk factor for the development of bladder cancer. Another crucial experience happened in 1955 when Hughes et al. described adverse drug reactions after therapy of tuberculosis patients using isonicotinic acid hydrazide (Hughes et al. 1955). In this time N-acetylation was identified to be responsible for individual drug response making it possible to differentiate between slow and rapid acetylators.  

Exposure to toxins like dioxin and other environmental chemicals have been shown to be metabolized by enzymes of phase I and/or phase II genes. In 1993 it was published by an expert team of the World Health Organization (WHO) that these enzymes are ‘biomarkers of susceptibility… which may increase or decrease an individual's risk of developing a toxic response following exposure to an environmental agent. Polymorphism is present for some metabolic activation/deactivation enzymes, including cytochrome P-450 isozymes and at least one form of glutathione transferase. Differing rates of enzyme activity controlling the activation or detoxification of xenobiotics lead to differences in susceptibility by increasing or decreasing the biologically effective dose of the environmental agent’ (WHO 1993).

In addition to cytochrome P450 and glutathione S-transferase the metabolic polymorphism of the N-acetyltransferase after low-level environmental exposure to carcinogens has been described to be genetically based (Vineis et al. 1994). Furthermore, several other phase II enzymes of the glutathione S-transferases have been reported to be involved in the detoxification of chemicals (Hallier et al. 1993; Hayes et al. 2000; Seidegard et al. 1997) which are able to modify the individual disposition to diseases of human beings. Taking all these observations together it is becoming obvious that genetic factors may influence the disposition for the development of chemical-induced syndromes like multiple chemical sensitivity syndromes (MCS).

According to Cullen (1987) the following criteria were used to definate the symptoms of multiple chemical sensitivity syndromes (MCS) (Table 1):

Table 1  MCS-Criteria

The prevalences of multiple chemical sensitivities in different populations are yet unknown. In a random sampling of 1.582 individuals from the Atlanta (Georgia) Caress and Steineman (2003) studied  the prevalence of multiple chemical sensitivities (MCS). They reported the hypersensitivity to common chemicals in 13.5% of the sample. They remarked that technological progresses within the last ten years have made it possible to introduce rapid and reliable tests for genotyping i.e. in the area of pharmacogenetic approaches (Weber 2001; Schmitz et al. 2001).  

A wide number of single nucleotide polymorphisms (SNPs) in the human genome has been identified so far. Several of these SNPs are located within phase I and phase II genes leading to an altered enzyme activity. The investigation of chemical-induced diseases is of importance because epidemiological studies have indicated that most human cancers are originally caused by long-term exposure to genotoxic agents. According to Doll and Peto (1981), 80 to 90 % of all cancers are related to environmental factors, tobacco smoke and diet. It is increasingly obvious that genetic differences among individuals in the ability to metabolize carcinogens like polycyclic aromatic hydrocarbons, aromatic amines, and nitroso compounds may play a primary role concerning the susceptibility to develop serious diseases like cancer (Idle 1991; Nebert 1991). The knowledge about the genetic relevance of metabolic variability has revealed new possibilities for studies focusing on increased susceptibility to environmental caused cancer and other environmental-influenced diseases.

Materials and Methods

The concept of this study was approved by the local ethic commission after pilot studies (Fabig 2000; 2002) to validate a questionaire for self reported sensitivites,  which was developed by Miller and Prihoda (1999). This questionnaire is a standardized approach for measuring chemical intolerances for research and clinical applications named QEESI (quick environmental exposure and sensitivity inventory).  All patients since Jan. 2000 - at the date 31.12.2003 2.746 individuals -  were offered to answer this questionnaire. The modified QEESI contains - like the US-original - fifty items about  quality, intensity, duration, localisation and modification of symptoms associated with environmental chemical exposure.

1.143 individuals answered this questionnaire without any medical influence or assistance.  One focus in this study was to analyze the scores of the QEESI-items, in which the individuals evaluate their feelings of the “sensitizing capabilities of chemicals”  (Table 2):

Table 2  The items and score conditions to measure the sensitizing capabilities of chemicals (SCC)

The chemical relatetd symptoms (CRS)  of the patients were head-related, muscle-related, neuromuscular, cognitive, gastrointestinal etc. (Table 3). The summaries of the CRS-scores of each individual fluctuate – analog to the SCC-Scores  - from zero (=no symptoms)  to 20 points (=maximum of symptoms)  were not shown in a later study.

Table 3  Chemical related symptoms (CRS)

861 of  1.143 persons gave an informed consent  for genotyping  enzymes of phase I and phase II in their xenobiotic metabolism. EDTA blood was sent to the molecularbiologist, who  isolated DNA from EDTA blood as described by Lahiri and Nürnberger (1991) or using QIAamp DNA Blood Mini Kit. Genotyping was performed in all patients at N-acetyltransferase 2 (NAT2), glutathione S-transferase M1 (GSTM1) and glutathione S-transferase T1 (GSTT1).  Dr. E. Schnakenberg described his part  as followed (personal communication): “After DNA extraction the N-acetyltransferase 2 gene was amplified as described previously (Schnakenberg et al. 2000). The single nucleotide polymorphisms (SNPs) nt 481, nt 590 and nt 857 of N-acetyltransferase gene were analysed in all individuals using RFLP or real-time PCR. According to the nomenclature of Vatsis et al. (1995) a simplified allele model was developed. The single nucleotide polymorphism nt 481 is a leading mutation which reflects the alleles NAT2*5A and NAT2*5B. The rare allele NAT2*5C was not identified by this procedure. These single nucleotide polymorphisms lead to a 4-allele model of the NAT2  which can predict the acetylator phenotype with an accuracy of more than 95 % for slow and rapid acetylation (Blum et al. 1991.)

The detection of the deletion of glutathione S-transferase gene M1 and/or T1 was performed by multiplex-PCR as described previously”.

The main substrates and the abbreviations  (used symbols in this study)  of  the  gene variants are shown  in table 4.

Table 4  Main substrates and used abbreviations  of  the  studied gene variants

343 individuals, which answered the questionnaire and 61 individuals with genotyping  were excluded from the study, because they were either no caucasians  or  at ages < 20 or > 90 years, or had a history of psychiatric and/or neurologic disease, which may be accused  as a confounder in studying MCS.

Data  and statistical analysis

Statistics were performed using the SPSS software version 10.0. Calculation of odds ratios with a confidence interval of 95 % was performed to analyse the associations between self-reported chemical sensitivity and single nucleotide polymorphisms. To assess the significance of associations Pearson correlation (chi square), Fisher’s exact test and logistic regression were used. 

Results  

Demographic data

The study group consists of 447 (56%) female and 353 male individuals. Age data  show a plateau at  the age from 40 to 70 years.  The mean age was 52.1  (± 14.6) and the median  52.6 years. The age groups and genders differed not significant (Chi-Quadrat-Test 0.07; figure 1).

Figure 1 age groups and gender  (N=800)

Genotyped data

Frequencies of genotyping the NAT2, GSTM1 and GSTT1 are shown in table 5.

Table 5 Frequencies of NAT2-, GSTM1- and GSTT1-gene variants

• The prevalence of slow N-acetylators in a reference-study of Cascorbi (et al. 1995) in Germany was 58.9%.  In accordance to these findings the frequency of slow acetylators in the present study was 58.1%.  
• GSTM1 gene deficience was detected in 53.5% of 416 white people of the US (Chen 1996). found this In the present study genotype  GSTM1 *0/*0 was found in the prevalence 52.2 percent.  
• Bruhn (et al. 1998) reported in a reference study (140 germans without illness)   the  deficience of the GSTT1 gene in 19.3 percent of all cases.   Smaller frequencies of this genotype   were analyzed  in white US-Americans: 14.7 percent by Wourmhoudt  (1999). In the current study frequency of GSTT1-Non-Conjugators  was  17.0 percent.

Summerizing these results one can say that  the study group is a very representative collective under the aim of studying the frequencies of these gene variants (Hirvonen 1993). Therefore also  the arithmetic combinations of these gene variants are representative for Caucasians (table 6).

Table 6  Frequencies of combinations of NAT2-, GSTM1- and GSTT1-gene variants (N=800)

Psychometric data

Figure 2 shows the frequencies of   the sum scores of  the reported sensitizing capabilities of  chemicals (SCC) the . The individual minimum score was zero  (= not at all a problem by chemical sensitizing), the individual maximum was a sum score of 20 points (=severe sensitizing by all of the asked chemicals).  The mean of all SCC scores was 9.5 points (± 5.6).

Figure 2 Frequency of sum scores of sensiziting capabilities of chemicals (N=800)

The  mean of the female SCC scores was 10.2 (± 5.6), the median 10.0.  Male had a non significant lower mean 8.6 (± 5.6) and a median of 9.0 (figure 3).

Figure 3 Gender and SCC-Scores

The generations lower than 40 years reported a lower capability of sensitizing by chemicals. The elderly (more than 70 years) did also. The linear regression of the SCC score with the body mass index showed no correlation  (R=0.006). Non-smokers reported  higher SCC scores than nicotine user (SCC mean 7.8 ± 5.1; median 8.0). They seemed to be more sensitive than smoker (SCC mean 10.3 ± 5.7; median 10.0; figure 4).

Figure 4 nicotine user and SCC scores (N=800)

Table 7  Sensitizing chemicals and answers (N=800)

How the  800 sum scores of the reported sensitizing capabilities of chemicals were related to the preformed classification MCS or not MCS (Cullen`s criteria) were analyzed with  logistic regression.

The results (numbers and percents) were shown in table 8.

Table 8  Results of logistic regression combining MCS and SCC scores (N=800)

The sensitivity of using  SCC scores in detecting MCS was 89.0 percent. The specifity of SCC-method was 87.4 percent.  In the ROC  (Receiver Operating Characteristics) curve sensivity and specifity were combined (figure 5).

Figure 5  MCS (Cullen`s criteria) and SCC score from modified QEESI (N=800)

Case control by combining the genotyped and the psychometric data

The relations between the dichotomizing SNPs and the  graduations in the questionnaires were analysed after excluding the answer “moderate symptoms”. The cases were 800  individuals with the answer “diasabling symptoms” from contacted chemicals. The controls were  randomized from the whole study group. The results in relations to NAT2, GSTM1 and GSTT1 variants are shown  in the tables 9a, 9b and 9c.

Table 9a  NAT2 and sensitizing by chemicals. Number of cases, randomized controls and OR.

Table 9b  GSTM1  and sensitizing by chemicals. Number of cases, randomized controls and OR.

Table 9c  GSTT1  and sensitizing by chemicals. Number of cases, randomized controls and OR.

Table 10  shows gene variants, cases  with SCC 10-20 or SCC 0-9  and odds ratios in individuals without  reference-sequence gene variants after randomizing the group of controls.

Table 10 Hypersensitivities (odds ratios) in individuals without reference-sequence gene variants.

All three odd ratios were higher than 1.0 and their  95% confidence intervalles  included never 1.0.

These data are shown in figure 6.

Figure 6  Odds ratios of hypersensitivity  by chemicals in individuals with either slow N-acetylisation or GSTM1- or GSTT1- genotype *0/*0.

The means of  800 SCC scores in relation to the combinations of the eight gene variants were shown in figure 7.

 Figure 7  Combinations of gene variants and means of SCC scores  (N=800)

The chemical sensitivity had the lowest score, when  gene variants of  the reference sequences were combined individuals. The sensitivities had the highest score , when the non reference sequence genotypes were combined. The GSTT1 genotype *0/*0  seemed  to be connected  with the more sensitive individuals (in background).

Medians and quartiles of the SCC score in relation to the combinations of gene variants demonstrate a similar picture (figure 8).

Figure 8 SCC scores  (box&whiskers) and combinations of  gene variants (N=800)

The prevalence of hypersensitivity in the background was 18.4 percent, hypersensitivity following elevated exposure was 32.9%  (Chi-Quadrat p < 0.000). 15.6% of the exposed were not sensitive against chemicals. 33.1 percent of the study group was neither exposed nor sensitive.

Table 11  Numbers and prevalences of exposures und sensitivities (N=800)

Conclusion

Hypersensitivities are caused by present or past exposure to chemicals either in the background or in special situations or conditions. A case control group with 800 individuals  reported individual different sensitivities and  documented the„sensitizing capabilities of chemicals “ (SCC). 2400  gene variants of NAT2, GSTM1 and GSTT1, which seemed to be probably three of the most important genes in xenobiotic metabolism, were  genotyped.

The results show different susceptibilities respect. their  outcome  different sensitivities, which are caused by different  specific genetic properties. The  highest odds ratio  of  hypersensitivity  (higher SCC score)  was related to GSTM1 genotype *0/*0 (OR 4.0).  OR of  GSTT1  genotype  *0/*0  was 1.8 (1.1-2.9). Risk of slow N-acetylators was 2.4 (1.7-3.9). Current (unpublished) data show that also the SNPs of the genes Paraoxonase 1 (PON1), Epoxidhydrolase (mEH) and Cytochromes (CYPs)  etc. are contributing factors in chemical sensitivities. 

But gene variants respective SNPs are not the sufficient cause of hypersensitivity. Gene variants are contributing aetiologic factors, which only modify the toxic effects of exposures. Elevated exposed people in the study had a prevalence of hypersensitivity of 32.9 percent. Prevalence  of hypersensitivity in background exposed individuals was 18.4 percent.

The analysed  gene variants have -  according to the analoge  structures studied in pharmacogenetics - an evolutionary relatively stable position. In former times they were generated to metabolize  toxicities and biochemics in nature life.   Living creatures in the last centuries or decades were more and more stressed by multiple anthrogenic substrates.  It seems alarming, that individuals in a common environment with “normal background” exposure and without diseases report more sensitizing effects of  chemicals, if they own certain combinations of  only three gene variants. Therefore seems that  REACH must not only regulate and evaluate  the  CMR substances (carcinogenic, mutagenic and reprotoxic) and the substances with POP characteristics (persistent organic pollutants) but also must establish the conditions of  specific permission (authorisation) of all chemicals. In addition we need characterizing  the neurotoxic properties of  lipophilic substrates, who are risk factors for developing  toxic enzephalopathy (TE). The risk for TE in solvent exposed men was elevated in the cases of GSTM1 gene deficience (Soderkvist 1996) 

Understanding hypersensitivity probably as a  unwanted product of former economics and risk managements the European REACH process needs the framework of genotyping and analyzing  the   sensitizing capabilities of  chemicals, before  Multiple Chemical Sensitivity Syndromes (MCS) grow from handicaps to diseases.

Address:

Karl-Rainer Fabig, Immenhöven 19, D-22417 Hamburg, Fax 049-40-53047272, e-mail  arztpraxis@fabig.org

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