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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:

CRS

Chemical related symptoms

CYP1A1

cytochrome 1A1

CYP1A2

cytochrome 1A2

CYP2D6

cytochrome 2D6

GSTM1

glutathione S-transferase M1

GSTP1

glutathione S-transferase P1

GSTT1

glutathione S-transferase T1

MCS

multiple chemical sensitivity syndrome

NAT2

N-acetyltransferase 2

PCR

polymerase chain reaction

PON1

paraoxonase 1

QEESI

quick environmental exposure and sensitivity inventory

RFLP

restriction fragment length polymorphism

SCC

sensitizing capability of chemicals

SCE

sister chromatid exchange

SNP

single nucleotide polymorphism

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):

MCS-Criteria  (Cullen 1987)

· it is acquired after a specific health event in association with an environmental exposure

· symptoms involve more than one organ system

· symptoms recur and abate in response to predictable stimuli

· symptoms are elicited by exposure to chemicals of diverse classes and modes of action

· symptoms occur in response to very low levels of chemicals

· no widely available test of organ system function can explain the symptoms.

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):

Please indicate

Not at all

Moderate

Disabling

whether or not these odors or exposures 

a problem

symptoms

symptoms

would make you feel sick …

(0)

(1)

(2)

  1. Diesel or gas engine exhaust

 

 

 

  1. Tobacco smoke

 

 

 

  1. Insecticide

 

 

 

  1. Gasoline

 

 

 

  1. Paint or paint thinner

 

 

 

  1. Cleaning products such as desinfectants, bleach,  bathroom cleaners or floor cleaners

 

 

 

  1. Certain perfumes, air fresheners or other fragances

 

 

 

  1. Fresh tar or asphalt

 

 

 

  1. Nailpolish, nailpolish remover, or hairspray

 

 

 

  1. New furnishings such as new carpeting, a new  soft plastik shower curtain or the interior of a new car

 

 

 

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.

Chemical-related symptoms (CRS)

  1. Musculoskeletal
  1. Airway-related
  1. Heart/chest-related
  1. Gastrointestinal
  1. Cognitive
  1. Affective
  1. Neuromuscular
  1. Head-related
  1. Skin-related
  1. Mucous membrane-related

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.

NAT 2

GSTM1

GSTT1

Typ. Substrate:

Benzidine

Typ. Substrate:

Benz(a)pyrene

Typ. Substrate:

Dichlorethane

substrate group:

aromatic

amines

Substrate group:

analoge substrates

Subtrate group:

Mono-Di-

Halo-methane

used symbols of gene variants

N0 : Slow acetylator

M0 : GSTM1- gene deficience

T0 : GSTT1- gene deficience

N1 : Rapid acetylator

M1 : GSTM1-reference-sequenz

T1 : GSTT1- reference-sequenz

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.

NAT 2 - rapid

N

percent

NAT2 - slow

N

percent

4/4

71

8.9

5/5

168

21.0

4/5

161

20.1

5/6

217

27.1

4/6

94

11.8

5/7

12

1.5

4/7

9

1.1

6/6

64

8.0

 

 

 

6/7

4

0.5

rapid N-acetylator

335

41.9

slow N-acetylator

465

58.1

GSTM1 *1/*1

380

47.5

GSTM1 *0/*0

420

52.5

GSTT1 *1/*1

664

83.0

GSTT1 *0/*0

136

17.0

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).

Combinations of gene variants

N

percent

N1*M1*T1

138

17.3

N1*M1*T0

23

2.9

N1*M0*T1

144

18.0

N1*M0*T0

30

3.8

N0*M1*T1

181

22.6

N0*M0*T1

202

25.3

N0*M1*T0

39

4.9

N0*M0*T0

43

5.4

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)

Sensitizing chemical or mixture

Not at all

a problem

Moderate

symptoms

Disabling

symptoms

  1. Diesel or gas engine exhaust

188

403

209

  1. Tobacco smoke

180

397

223

  1. Insecticide

258

334

208

  1. Gasoline

196

377

227

  1. Paint or paint thinner

143

343

314

  1. Cleaning products such as desinfectants, bleach,  bathroom cleaners or floor cleaners

292

346

162

  1. Certain perfumes, air fresheners or other fragances

257

340

203

  1. Fresh tar or asphalt

328

339

133

  1. Nailpolish, nailpolish remover, or hairspray

253

355

192

  1. New furnishings such as new carpeting, a new  soft plastik shower curtain or the interior of a new car

351

287

162

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.

 

Predicted

 

 

Observed

MCS

No MCS

Percent correct

 

MCS

365

45

89.0

 

No MCS

49

341

87.4

 

total

 

 

88.3

 

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.

N-acetyltransferase  gene variants

Disabling symptoms

Not at all a problem

 slow acetylator

Sensitizing  chemical or mixture

N0

N1

sum

N0

N1

sum

OR

95%-CI

  1. Diesel or gas engine exhaust

137

72

209

40

59