Toxicogenomics of subchronic hexachlorobenzene exposure in Brown Norway rats.

Hexachlorobenzene (HCB) is a persistent environmental pollutant with toxic effects in man and rat. Reported adverse effects are hepatic porphyria, neurotoxicity, and adverse effects on the reproductive and immune system. To obtain more insight into HCB-induced mechanisms of toxicity, we studied gene expression levels using DNA microarrays. For 4 weeks, Brown Norway rats were fed a diet supplemented with 0, 150, or 450 mg HCB/kg. Spleen, mesenteric lymph nodes (MLN), thymus, blood, liver, and kidney were collected and analyzed using the Affymetrix rat RGU-34A GeneChip microarray. Most significant (p < 0.001) changes, compared to the control group, occurred in spleen, followed by liver, kidney, blood, and MLN, but only a few genes were affected in thymus. This was to be expected, as the thymus is not a target organ of HCB. Transcriptome profiles confirmed known effects of HCB such as stimulatory effects on the immune system and induction of enzymes involved in drug metabolism, porphyria, and the reproductive system. In line with previous histopathological findings were increased transcript levels of markers for granulocytes and macrophages. New findings include the upregulation of genes encoding proinllammatory cytokines, antioxidants, acute phase proteins, mast cell markers, complements, chemokines, and cell adhesion molecules. Generally, gene expression data provide evidence that HCB induces a systemic inflammatory response, accompanied by oxidative stress and an acute phase response. In conclusion, this study confirms previously observed (immuno)toxicological effects of HCB but also reveals several new and mechanistically relevant gene products. Thus, transcriptome profiles can be used as markers for several of the processes that occur after HCB exposure. Key words: Brown Norway rat, DNA microarray analysis, drug metabolism, estrogen metabolism, genomics, hexachlorobenzene, immunotoxicity, inflammation, oxidative stress, porphyria. Environ Health Perspect 112:782-791 (2004). doi:10.1289/txg.6861 available via http://dx.doi.org/[Online 7 April 2004]

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Hexachlorobenzene (HCB) was used as a fungicide until the 1970s, when such use was prohibited. Considerable amounts are still generated as waste by-products of industrial processes and emitted into the environment. Because of its chemical stability, persistence, and long-range transport, HCB can be found throughout the environment and is detectable in human milk, blood, and adipose tissue.

In the 1950s, an accidental poisoning in Turkey revealed several toxic effects of HCB in humans. Approximately 3,000-5,000 people ingested HCB-treated seed grain and developed a disease called porphyria turcica (Gocmen et al. 1986), characterized by hepatic porphyria and cutaneous skin lesions caused by a disturbed porphyrin metabolism (Bickers 1987). Other clinical symptoms include enlarged liver, spleen, lymph nodes (LNs), and thyroid, neurological symptoms, and arthritis. Infants born to mothers exposed to HCB developed a different syndrome called pembe yarn, characterized by high mortality, diarrhea, fever, hepatomegaly, and skin lesions in the absence of porphyria, but with infiltrations of macrophages and lymphocytes and infiltrates in the lung (Cam 1960). Immunotoxic effects were reported in the Turkish poisoning victims, but also in occupationally exposed workers in Brazil. Increased levels of IgM and IgG were observed, as well as impaired function of neutrophil granulocytes (Queiroz et al. 1998a, 1998b).

In rats HCB induced hepatic porphyria and neurotoxic effects (Courtney 1979), and toxic effects on the reproductive system (Jarrell et al. 1998), thyroid function (Kleiman de Pisarev et al. 1990), and immune system (Michielsen et al. 1999; Vos 1986). Because HCB is a lipophilic xenobiotic, exposure leads to accumulation in adipose tissue, whereas only a small part of ingested HCB is metabolized. HCB can be converted in a cytochrome P450 (CYP)-dependent manner (Van Ommen and Van Bladeren 1989) and also via the mercapturic acid pathway (Renner 1981).

Brown Norway (BN) rats are very susceptible to HCB-induced adverse immune effects. Exposure caused a dose-dependent immunostimulation characterized by enlarged spleen and LNs and increased serum levels of total IgM, IgG, IgE, and IgM against single-stranded (ss)DNA. Furthermore, rats developed inflammatory skin and lung effects characterized by infiltrates of eosinophilic granulocytes and macrophages (Michielsen et al. 1997, 1999). Although both T cells and macrophages seem to play an important role in HCB-induced immunotoxicity in BN rats (Ezendam et al. 2004), exact mechanisms are unknown.

In this study we used DNA microarray analysis to assess changes associated with HCB exposure at the gene expression level. Transcript levels were measured using the Affymetrix RG U34A GeneChip. BN rats were exposed to 0, 150, or 450 mg HCB per kg diet, doses used also in earlier studies (Ezendam et al. 2004; Michielsen et al. 1997), and gene expression levels were assessed in spleen, mesenteric lymph nodes (MLN), thymus, blood, liver, and kidney. This approach revealed several changes in line with the known toxic effects but also revealed novel ones, which may suggest additional (immuno)toxic effects of HCB exposure and/or provide more insight into the mechanisms of HCB-induced adverse effects.

Materials and Methods

Rats and Maintenance

Three-week-old SPF female inbred Brown Norway (BN/SsNO1aHsD, termed BN) rats were purchased from Harlan (Blackthorn, UK). Rats were acclimatized for 1 week before the start of the experiment. They were kept two by two under standard conditions with food and acidified drinking water ad libitum. The diet consisted of a semisynthetic diet (SSP/TOX; Hope Farms, Woerden, the Netherlands) with or without crystalline HCB (99% purity; Aldrich Chemie, Bornem, Belgium) by mixing of homogeneity. The experiments were approved by the animal experiments committee of the Faculty of Veterinary Medicine of the Utrecht University.

Experimental Protocol

Rats were randomly assigned to different experimental groups (n = 6) receiving either control diet or the diet supplemented with 150 mg (low dose) or 450 mg (high dose) HCB/kg. Body weight (bw) and skin lesions were recorded twice per week. After 28 days rats were killed by C[O.sub.2]/[O.sub.2]. Blood was collected in tubes containing EDTA to prevent clotting and transferred into Fastubes (Endotell, Allschwill, Switzerland) containing guanidinium isothiocyanate in 0.9% NaCl solution. Tubes were snap-frozen in liquid nitrogen. Spleen, MLN, thymus (freed from adjacent LN), liver, and kidney were collected, weighed, and snap-frozen in liquid nitrogen.

In additional experiments for pathology, blood, and serum analysis, rats were exposed to the same dosing regimens. Rats were killed by a lethal dose of pentobarbital (Euthesate; 0.3 g/kg bw ip; Ceva Sante Animal B.V., Maassluis, the Netherlands). One part of the blood was collected in EDTA tubes for total and differential leukocyte counts; the other part was used for serum analysis. Spleen, MLN, thymus, liver, and kidney were fixed in phosphate-buffered 4% formaldehyde; after embedding in Paraplast, 5-[micro]m sections were stained with hematoxylin and eosin.

DNA microarray experiment. Total RNA was obtained by acid guanidinium isothiocyanate-phenol-chloroform extraction (Trizol; Invitrogen Life Technologies, San Diego, CA, USA) (Chomczynski and Sacchi 1987) and purified on an affinity resin (RNeasy; Qiagen, Hilden, Germany) according to manufacturer instructions. DNA microarray experiments were conducted as recommended by the manufacturer of the GeneChip system (Affymetrix, Inc. 2002) and as previously described (Lockhart et al. 1996). Rat specific RG U34A gene expression probe arrays (Affymetrix, Inc., Santa Clara, CA, USA) were used containing 8,799 probe sets interrogating primarily annotated genes. Per tissue and per animal, one chip was used. The resulting image files (.dat files) were processed using the Microarray Analysis Suite 5 (MAS5) software (Affymetrix, Inc.). Tab-delimited files were obtained containing data regarding signal intensity (Signal) and categorical expression level measurement (Absolute Call).

Data Analysis

To determine which genes were diffentially expressed between the three treatment groups, a one-way analysis of variance (ANOVA) was applied to genes that had a present call in at least one of the samples. Genes with a p-value < 0.001 were considered statistically significant. Group average fold changes were calculated by using the average of the low- or high-dose groups compared with the control group. The annotation of the genes was determined by using NetAffx (http://www.affymetrix.com; Liu et al. 2003). Further information on probe sets was found in the literature or in the KEGG database (http://www.genome.ad.jp/kegg/kegg2.html). Additional data analysis by principal component analysis (PCA) was performed using GeneMaths (Applied Maths, Sint-Martens-Latem, Belgium). Averages of gene expression levels in control, low-, and high-dose groups were calculated; low values were cut off using a lower threshold of 10, and the values were log transformed before PCA.

GC-MS Analysis of Contamination in the Hexachlorobenzene Sample

To analyze HCB for contaminating polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), a solution of acetone containing [sup.13]C12-labeled internal quantitation standards (Cambridge Isotope Laboratories, Woburn, MA, USA) of the PCDDs and PCDFs was added to dichloromethane. The solution was brought to a Carbosphere (Alltech B.V., Zaandam, the Netherlands) column, then purified on [Al.sub.2][O.sub.3], evaporated to dryness, and redissolved in toluene. Gas chromatography-mass spectrometry (GC-MS) analyses were performed on a double-focusing mass spectrometer coupled to a gas chromatograph. GC separations were carried out on a nonpolar capillary column (60 m DB-5MS; 0.25 mm ID, 0.10-[micro]m film thickness; J&W Scientific, Folsom, CA, USA). Ionization of the sample was performed in the electron impact mode. Detection was performed by selected ion recording.

Results and Discussion

Body Weight Gain, Macroscopic Skin Lesions, and Organ Weights

During treatment with the low-dose diet, body weight increased significantly from day 10 onward, whereas rats exposed to the high-dose diet had a significantly higher body weight on days 10 and 20 (data not shown). One of the rats in the high-dose group died after 25 days of exposure to HCB. Time of onset, severity, and size of the skin lesions were similar as described previously (Michielsen et al. 1997). Increased liver and spleen weights in both dosing groups were also in accordance with previous work, as were the observed histopathological changes in these organs (Michielsen et al. 1997). In the high-dose group, kidney weight increased significantly, as observed before in Wistar rats treated with HCB for 25 days (Kennedy and Wigfield 1990) but not in BN rats treated with HCB for 21 days (Michielsen et al. 2002). Histopathological changes were not observed. Thymus weight decreased significantly in the high-dose group. It is likely that this thymus atrophy is caused by stress, as typical stress-induced alterations (Kuper et al. 2002) were observed. No significant differences in MLN weight were found, but histopathology of MLN of the high-dose group showed comparable morphology as reported previously (Michielsen et al. 1997).

DNA Microarray Analysis

The PCA plot (Figure 1) of the ratios of the low- and high-dose groups over the control group shows that gene expression in spleen, blood, and liver is dose dependently changed, whereas this is less clear for MLN, thymus, and kidney. Spleen and blood cluster close together, as do kidney and thymus, but liver and MLN are more distant from those tissues. Most significant changes (p < 0.001) in gene expression occurred in spleen (679 probe sets), followed by liver (346), kidney (232), blood (144), MLN (104), and thymus (28). The low number of changes in thymus is not surprising, as the thymus is not a target organ of HCB. Remarkably in kidney, many genes were affected, although this organ has rarely been described to be affected by HCB. Furthermore, although organ weights were increased, no histopathological changes were detected in the present study. Because not all significantly changed genes can be included in this article, we present only genes associated with immunology (Tables 1-6), acute phase responses (APRs) and oxidative stress (Table 7), and enzymes involved in drug metabolism, porphyria, and estrogen metabolism (Table 8).

[FIGURE 1 OMITTED]

Figure 2 shows a deduced scheme of immune cells and mediators involved in the inflammatory response. This scheme is used to simplify the cascade of reactions that occur during inflammation and to present the results in a logical order. The complete list of significantly changed probe sets can be found on the ArrayExpress website (http://www.ebi.ac.uk/arrayexpress).

[FIGURE 2 OMITTED]

Inflammatory Response

Macrophages. In HCB-exposed rats, macrophage infiltrations were observed in skin, lung (Michielsen et al. 1997), spleen (Ezendam et al. 2004; Schielen et al. 1993), and liver (Courtney 1979). As expected, HCB increased gene expression of macrophage markers in spleen and MLN and Kupffer cell markers in liver, supporting the significance of macrophages in HCB-induced immunotoxicity.

Proinflammatory cytokines. Gene expression of the receptor for tumor necrosis factor (TNF)[alpha] and TNF[beta] (TNF receptor superfamily, member 1) in MLN, spleen, and kidney was increased. In addition, IL-6 gene expression was affected in MLN, just as the IL-6 signal transducer in kidney. IL-6 is a pleiotropic cytokine that plays an important role in B-cell differentiation, growth of T cells, and differentiation of macrophages (Naka et al. 2002). HCB also induced gene expression of IL-1[beta] in spleen (low-dose group) and IL-1[beta]-converting enzyme in kidney, an enzyme that converts IL-1[beta] and IL-18 to their active form. Gene expression of IL-18, a cytokine produced mainly by Kupffer cells, was elevated in liver.

p38 MAPK signaling pathway. The mitogen-activated protein kinase (MAPK) family consists of signal transduction molecules important during inflammation. HCB induced expression of p38 MAPK and other MAPKs in kidney. Activation of p38 MAPK leads to phosphorylation of several transcription factors, such as signal transducer and activator of transcription-1 (STAT-1). Gene expression of STAT-1 was increased in liver. Both MAPK and STAT-1 are important in cytokine production, and negative regulation of cytokine signaling occurs at the level of transcription of these molecules. Proteins involved in suppression of cytokine production are the so-called suppressors of cytokine signaling (SOCSs). HCB exposure increased gene expression of several of these proteins, probably to counteract the high cytokine levels. In spleen, SOCS-2 was upregulated in the low-dose group, but downregulated in the high-dose group, and SOCS-3 was upregulated in MLN. In the thymus, cytokine inducible SH2-containing protein was upregulated, a protein that plays a critical role in controlling T-cell activation (Chen et al. 2003).

Oxidative stress and antioxidants. Previous studies have shown that HCB exposure induced oxidative stress (Billi de Catabbi et al. 1997) and increased expression of antioxidants in the liver (Stonard et al. 1998). The present work confirms these findings, as several antioxidants were induced in liver. Transcriptome profiles show that antioxidants are also increased in spleen, MLN, blood, and kidney. The infiltrated macrophages and granulocytes probably generate these reactive oxygen species (ROS). Additional experiments showed that serum hydroperoxides were significantly increased in HCB-exposed BN rats (data not shown). Excessive presence of ROS can activate nuclear factor kappa B, an important factor in regulating the inflammatory response (Schreck et al. 1992). In addition, ROS can cause cell damage, providing danger signals that can attract inflammatory cells. Therefore, increased oxidative stress induced by HCB may play a pivotal role in the observed immunostimulation.

Acute phase response. Acute phase proteins (APPs) are important in inflammatory responses. HCB increased gene expression of several APPS, such as heat shock proteins (HSPs) in spleen and MLN. HSPs protect cells against cellular stress. HCB also increased gene expression of matrix metalloproteinase-9 (MMP-9) in spleen and of the natural inhibitors of MMPs, tissue inhibitor of metalloproteinase-1 (TIMP-1) in liver and TIMP-2 in MLN. MMPs play an important role in the cleavage of membrane components, enabling leukocytes to extravasate the blood. HCB also affected transcript levels of other APPs, such as haptoglobin (a hemoglobin scavenger), lipopolysaccharide-binding protein, orosomucoid (important in immunomodulation), and metallothionein and ceruloplasmin (antioxidants). Negative APPs (transferrin and its receptor) were also induced; these proteins are normally down-regulated during an APR. Synthesis of these APPs, however, is also dependent on iron metabolism. HCB induced iron accumulation in the liver (Stonard et al. 1998). The upregulation of transferrin gene expression in spleen and kidney suggests that this is also the case in these organs.

Complement system. Complement components are also important in inflammatory responses. HCB increased gene expression of several components of the complement pathway in spleen, blood, kidney, and liver.

Mast cells. HCB enhanced gene expression of mast cell enzymes, probably a consequence of complement activation. This finding may also be explained by a characteristic of the BN rat, a strain that tends to respond in a more T helper-2--skewed fashion. Basal levels of serum IgE are high, and HCB increases IgE levels even more (Michielsen et al. 1997). Loading of mast cells with IgE may result in degranulation and release of inflammatory mediators.

Chemokines and chemokine receptors. In all analyzed organs, HCB increased gene expression of chemokines, important mediators in the recruitment of leukocytes from the circulation. HCB induced gene expression of several CXC chemokines and their receptors: lipopolysaccharide-induced CXC chemokine (LIX), chemokine (CXC motif) ligand 10, growth-related oncogene (Gro) and the CXC chemokine receptor 2 (CXCR2). LIX is a potent neutrophil chemoattractant, whereas chemokine (CXC motif) ligand 10 plays an important role in chemotaxis of activated T cells and monocytes. Gro is a ligand that binds to CXCR2, a receptor present on neutrophils. HCB induced gene expression of two CC chemokine receptors: CC chemokine-binding receptor JAB61, a receptor that binds monocyte chemoattractant protein-1 and -3, and the receptor for macrophage inflammatory protein-1[alpha] that is present on neutrophils and eosinophils (Mantovani et al. 1998).

Cell adhesion molecules. Chemokines induce expression of cell adhesion molecules on both endothelial cells and leukocytes. HCB affected gene expression of cell adhesion molecules in all organs except the thymus. Intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and selectin are endothelial cell adhesion molecules that recognize receptors on hemopoietic cells. Other cell adhesion molecules in which gene expression was induced by HCB were fibronectin-1, embigin, CD36, and glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1). The latter is expressed only on high endothelial venules (HEVs) in LNs. Previous reports have shown that HCB increased the development of HEVs in LNs (Michielsen et al. 1997), which probably results in increased GlyCAM-1 mRNA expression.

Granulocytes. Upregulation of chemokines and cell adhesion molecules leads to influx of leukocytes. Data obtained in this study confirm increased numbers of monocytes and neutrophilic granulocytes in blood (unpublished data) and cellular infiltrations in spleen of BN rats (Michielsen et al. 1999). In all analyzed organs and blood, gene expressions for S100 calcium-binding protein A8 (MRP-8) and A9 (MRP-14) were upregulated. These proteins are abundantly present in the cytoplasm of neutrophils, monocytes, and macrophages (Roth et al. 2003). Other markers associated with granulocytes and macrophages that were affected by HCB were defensin (neutrophils and macrophages), lipocalin (granulocytes), and CD24 (granulocytes, monocytes, and lymphocytes). HCB also induced gene expression of 12-lipoxygenase- and arachidonate 5-lipoxygenase-activating protein, both involved in leukotriene activation, which takes place in myeloid cells (Bigby 2002). Gene expression of Fc receptors was also elevated by HCB, probably because of the increase in the number of cells bearing this receptor. The same is true for the upregulation of gene expression of several pattern recognition molecules, such as CD14, mannose-binding lectin, and peptidoglycan recognition molecules, present on monocytes, macrophages, and neutrophils.

This work indicates that HCB exposure results in a systemic inflammatory response. To counterbalance this response, the immune system produces anti-inflammatory mediators. HCB exposure induced gene expression of one of these mediators, annexin-1, which blocks leukocyte migration and induces apoptosis in inflammatory cells (Perretti and Gavins 2003).

T and B Cells and Major Histocompatibility Complex II Expression

Gene expression of T-cell markers such as CD3 a subunit of the T-cell receptor, was decreased in spleen, whereas in blood, HCB decreased gene expression for CD3 and CD37, the latter being a B-cell marker. Furthermore, HCB increased gene expression of CD52 or B7 antigen, a marker present on antigen-presenting cells, such as B cells and monocytes. This is in line with previous studies that have shown a stronger increase of monocytes and granulocytes in blood after HCB exposure, resulting in relatively fewer lymphocytes (Schulte et al. 2002; Vos et al. 1979). In kidney we observed an increased expression of OX 45 (homolog to CD2), a membrane protein involved in the binding to LFA-3, important in adhesion of T cells to other cell types and in T-cell activation. HCB enhanced gene expression of immunoglobulins in spleen, MLN, liver, and kidney. This is in line with the observed increase of serum levels of IgM, IgG, and IgE in BN rats (Michielsen et al. 1997). Major histocompatibility complex (MHC)II gene expression was decreased in spleen and blood and increased in liver and kidney.

Autoantibodies

The anti-acetylcholine receptor antibody gene (rearranged Ig [gamma]-2a chain) was upregulated in spleen, thymus, liver, and kidney. These autoantibodies are associated with the autoimmune disease myasthenia gravis (MG), a neurological disease characterized by degeneration of the acetylcholine receptor and resulting in muscle weakness (De Baets and Stassen 2002). HCB-induced neurological effects, however, are not the same as symptoms described for MG. Additional experiments performed to detect antiacetylcholine receptors antibodies (total Ig) in serum did not confirm gene expression data. HCB exposure also increased gene expression of anti-nerve growth factor-30 antibodies in spleen and liver and downregulated expression in blood. These antibodies belong to the naturally occurring autoantibodies and are elevated in inflammatory diseases (Dicou et al. 1996). The exact role of these auto-antibodies is not yet known. Previously it was shown that HCB increased IgM antibodies against autoantigens such as ssDNA (Michielsen et al. 1997; Schielen et al. 1993). Expression of La (= autoantigen SS-B/La) was induced in kidney. This protein plays a role in RNA polymerization and is often a target of autoantibodies found in several autoimmune diseases (Huhn et al. 1997).

Drug-Metabolizing Enzymes

Cytochrome P450. CYP enzymes are involved in the oxidative dehalogenation of HCB (Van Ommen and Van Bladeren 1989). HCB exposure increased gene expression of several CYPs and of epoxide hydrolase, an enzyme involved in detoxification of epoxides in liver (Table 8). In spleen, MLN and kidney expression of CYP enzymes was also induced but to a lesser extent than in liver.

Role of dioxin-like contamination of HCB. Surprisingly, gene expression of CYP1A1 was strongly upregulated in liver. This was an unexpected finding, as previous work showed that HCB induced much more CYP2B than CYP1A1 (Franklin et al. 1997). CYP1A1 upregulation is associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or related compounds that activate the aryl hydrocarbon (Ah) receptor. It is still the subject of debate if HCB is a dioxin-like compound. Van Birgelen (1998) suggested that HCB should be considered as one, as HCB meets the criteria for dioxin-like compounds: the ability to bind to the Ah receptor, induction of dioxin-like effects, and bioaccumulation. Vos (2000) commented, however, that although TCDD and HCB share some target organs, the toxic effects in these systems are quite different. Furthermore the affinity for the Ah receptor is 10,000 times less for HCB than for TCDD (Hahn et al. 1989). HCB was analyzed to investigate whether contamination with dioxin-like compounds was responsible for the observed effects. Indeed, HCB was contaminated with PCDDs and PCDFs, and the toxic equivalent was 187 pg/mg HCB. The calculated no observed adverse effect level (NOAEL) of CYP1A1 induction was 0.7-4 ng TCDD/kg bw/day (Van Birgelen et al. 1995). In our study rats were exposed to approximately 2 ng/kg bw/day (low dose) and 6 ng/kg bw/day (high dose). Therefore, exposure to dioxins and furans is of the same order of magnitude as the calculated NOAEL and therefore not likely to be responsible for the observed strong increase in gene expression for CYP1A1. This is not in accordance with previous work showing that HCB could only moderately or not at all induce CYP1A1 by HCB (Franklin et al. 1997; Machala et al. 1996). This discrepancy may be explained by strain differences or by the difference in detection of CYP1A1 (7-ethoxy-resorufin-O-deethylase induction versus gene expression).

Mercapturic acid pathway. The BN rat degrades HCB also via the mercapturic acid pathway that involves glutathione conjugation catalyzed by glutathione S-transferase (GST; Renner 1981). As expected, gene expression of several GSTs was upregulated in liver. Other phase II enzymes that were induced are mercaptopyruvate sulfurtransferase, uridine diphosphate (UDP)-glucuronosyltransferase, and the sulfotransferase family.

Porphyria

One of the main toxic effects of HCB is the induction of porphyria in humans (Gocmen et al. 1986) and experimental animals (Courtney 1979), caused by a disturbance in heme biosynthesis. In the present study, gene expression of enzymes involved in heme synthesis were induced. These include aminolevulinate (ALA) dehydratase, porphobilinogen deaminase (hydroxymethylbilane synthase), and uroporphyrinogen decarboxylase in spleen and ALA synthase in liver.

Estrogen/Androgen Metabolism

Several reports have shown that HCB exposure induces effects on the reproductive system. In humans, serum HCB levels from women exposed during the accident in Turkey correlated with spontaneous abortion (Jarrell et al. 1998), and the proportion of male births was reduced in the group of women that had HCB-induced porphyria (Jarrell et al. 2002). In monkeys, HCB decreased estrogen levels (Foster et al. 1995), and in Wistar rats, HCB exposure reduced serum levels of estrogen and decreased levels of uterine estrogen receptors (Alvarez et al. 2000). Gene expression of estrogen sulfotransferase was upregulated in liver. This enzyme is important in the sulfation of estrogen, a pathway that inactivates estrogen. The enzyme 17[beta]-hydroxysteroid dehydrogenase was downregutated in the liver. This enzyme catalyzes the interconversion of testosterone and androstenedione as well as estradiol and estrone. Both can lead to lower estrogen levels. Together, these results indicate that HCB interferes with estrogen metabolism.

Conclusions

Gene expression profiles confirmed known effects of HCB such as stimulatory effects on the immune system and induction of enzymes involved in drug metabolism, porphyria, and the reproductive system. New findings include upregulation of genes encoding proinflammatory cytokines, antioxidants, APPs, complement, mast cell markers, chemokines, and cell adhesion molecules. Thus, most transcriptome profiles are consistent with and complementary to previous pathological findings and can be used as markers for several processes that occur after HCB exposure.

Presumably, after oral exposure to HCB, macrophages are attracted to organs such as spleen, lung, and skin and become activated by HCB. This leads to a cascade of reactions involving innate immune cells, as depicted in Figure 2. The gene expression profiles provide evidence for the importance of macrophages and granulocytes and mediators released by these cells in the adverse inflammatory response against HCB. In this way, co-stimulatory or danger signals are generated that could polyclonally activate T cells. Thus, DNA microarray analysis revealed the complexity of cells and mediators involved in the immune response elicited by HCB and confirms previous work showing the importance of macrophages and granulocytes (Ezendam et al. 2004; Michielsen et al. 1999).

Data obtained in an extensive study such as this can be used to create a database with gene expression profiles of known toxicants, as has been suggested previously (Thomas et al. 2002). Chemicals can be screened by establishing their gene expression profiles and comparing them with profiles of known toxic chemicals. In this way classes of toxic compounds can be recognized, as has previously been shown for hepatotoxicants (Hamadeh et al. 2002a, 2002b), and genomics may be an additional tool in hazard identification.

Table 1. Spleen: representative genes that changed significantly
(p < 0.001) after HCB treatment--immune system. (a)

                                                        Fold change

                                                   HCB low   HCB high
Accession number             Gene name               dose      dose

Granulocytes and
    macrophages
  AA957003         S100 calcium binding protein       2.8       34
                     A8
  U50353           Defensin 3a                        2.5       32
  AA946503         Lipocalin 2                        1.7       24
  L18948           S100 calcium binding protein       3.2       19
                     A9
  L06040           12-lipoxygenase                    1.9        5.7
  M32062           Fc receptor, IgG, low              1.4        2.0
                     affinity III
  AA894004         ESTs, highly similar to Capg       1.2        1.4
                     mouse macrophage capping
                     protein
  X73579           Fc receptor, IgE, low             -1.1       -2.3
                     affinity II

Mast cells
  U67913           Mast cell protease 10             12         42
  U67888           Mast cell protease 3               3.4       20
  U67907           Mast cell protease 4               1.5        8.7
                     precursor
  M21622           High-affinity IgE receptor         3.2        7.0
  U67914           Mast cell carboxypeptidase A       1.8        6.8
                     precursor
  U67908           Mast cell protease 5               1.2        6.0
                     precursor
  M38759           Histidine decarboxylase            3.7        4.3

Pattern
    recognition
    molecules
  AF087943         CD14 antigen                       1.1        1.7
  Complement
  AF036548         Response gene to complement       -1.3       20
  AA818025         CD59 antigen precursor             1.1        1.7

Cell adhesion
    molecules
  X05834            Fibronectin 1                     1.8        3.5
  AJ009698          Embigin                           1.4        3.3

Chemokines
  U90448           CXC chemokine LIX                  1.0        1.9
  U17035           Chemokine (CXC motif) ligand       1.0       -2.3
                     10

Cytokines and
    cytokine-
    associated
    genes
  M63122           Tumor necrosis factor              1.3        1.3
                     receptor
  AF075382         Suppression of cytokine            1.3       -1.3
                     signaling
  M98820           Interleukin 1 beta                 1.2       -1.2
  M55050           Interleukin 2 receptor beta        1.2       -1.4
                     chain
  L00981           Lymphotoxin, tumor necrosis       -1.1       -1.4
                     factor alpha
  M34253           Interferon regulatory             -1.1       -1.6
                     factor 1
  U14647           Interleukin 1 beta converting      1.1       -1.6
                     enzyme
  U69272           Interleukin 15                    -1.1       -1.7
  U48596           MAPK kinase kinase 1               1.0       -1.8
  U03491           Transforming growth factor,       -2.9       -3.0
                     beta 3

Genes associated
    with T and B
    cells and
    MHCII
    expression
  U39609           Anti-nerve growth factor 30        1.3        3.8
                     antibody light-chain
  L22654           Antiacetylcholine receptor         3.2        1.6
                     antibody rearranged
                     immunoglobulin gamma-2a
                     chain
  L07398           Immunoglobulin rearranged          1.0        2.4
                     gamma-chain V region
  M18526           Immunoglobulin germline            1.2        1.6
                     kappa-chain
  X13016           MRC OX-45 surface antigen          1.1       -1.3
  U11681           Rapamycin and FKBP12 target-1     -1.0       -1.3
                     protein
  D13555           T-cell receptor CD3, subunit      -1.1       -1.4
                     zeta
  U31599           MHC class II-like beta chain      -1.0       -1.4
                     RT1.Mb
  L14004           Polymeric immunoglobulin           1.0       -1.4
                     receptor
  D10728           Lymphocyte antigen CD5            -1.2       -1.6
  M85193           RT6.2                             -1.3       -1.6
  U24652           Linker of T-cell receptor         -1.0       -1.7
                     pathways
  X14319           T-cell receptor active            -1.2       -2.1
                     beta-chain, V region

EST, expressed sequence tag.

(a) Table contains GenBank accession numbers
(http://www.ncbi.nih.gov/entrez/query.fcgi?db=nucleotide) of
the cDNA fragments present on Affymetrix RG U34A gene chips, gene
name, and average fold change in expression of both low dose data from
five to six rats per group. A one-way and high dose versus control.
Fold changes are calculated with ANOVA was used to determine
significance; only probe sets that changed significantly with p <
0.001 are shown.

Table 2. MLN: representative genes that changed significantly
(p < 0.001) after HCB treatment--immune system. (a)

                                                      Fold change

                                                   HCB low   HCB high
Accession number             Gene name               dose      dose

Granulocytes and
    macrophages
  L18948           S100 calcium binding protein      2.2       22
                     A9
  AA957003         S100 calcium binding protein      2.6       19
                     A8
  M32062           Fc gamma receptor                 2.0        2.8
  AJ223184         DORA protein (immunoglobulin      1.1        2.6
                     superfamily, member 6)

Pattern
    recognition
    molecules
  U44129           Mannose-binding lectin 1          1.5        2.6
  AF087943         CD14 antigen                      1.8        2.5

Cell adhesion
  L08100           Glycam 1                          3.1        2.5

Chemokines
  U92803           CC-chemokine-binding receptor     1.9        2.6
                     JAB61
  AF053312         CC chemokine ST38 precursor       2.4       16

Cytokines
  M26744           Interleukin 6                     2.3        4.3
  AF075383         Suppressor of cytokine            1.9        2.5
                     signaling
  M63122           Tumor necrosis factor             1.2        1.8
                     receptor
  AA891209         ESTs, highly similar to           1.2        1.5
                     interleukin 25

Genes associated
    with T and B
    cells and
    MHCII
    expression
  M28671           Rearranged IgG-2b                 1.5        3.2
  X07189           Immunoglobulin heavy chain        2.5        3.1
                     constant region
  M18526           Immunoglobulin germline           1.4        1.8
                     kappa-chain

(a) Table contains GenBank accession numbers (http://www.ncbi.nih.gov/
entrez/quary.fcgi?db=nucleotide) of cDNA fragments present on
Affymetrix RG U34A gene chips, gene name, and average fold change in
expression of both law dose and high dose versus control. Fold changes
are calculated with data from five to six rats per group. One-way
ANOVA was used to determine significance; only probe sets that changed
significantly with p < 0.001 are shown.

Table 3. Thymus: representative genes that changed significantly
(p < 0.001) after HCB treatment--immune system. (a)

                                                        Fold change

                                                   HCB low   HCB high
Accession number             Gene name               dose      dose

Granulocytes and
    macrophages
  L18948           S100 calcium binding protein      1.1        2.0
                     A9 (MRP-14)
  X14323           IgG receptor FcRn                 1.2        1.2

Mast cell
  U67911           Mast cell protease 8              1.5        2.0
                     precursor

Cytokine
  AF065161         Cytokine inducible                1.2        1.7
                     SH2-containing protein

Genes associated
    with B cells
  L22654           Antiacetylcholine receptor
                     antibody rearranged immuno-     1.6        3.7
                     globulin gamma-2a chain,
                     VDJC region
  M18526           Ig germline kappa-chain           2.0        3.2

(a) Table contains GenBank accession numbers (http://www.ncbi.nih.gov/
entrez/query.fcgi?db=nucleotide) of cDNA fragments present on
Affymetrix RG U34A gene chips, gene name, and average fold change
in expression of both low dose and high dose versus control. Fold
changes are calculated with data from five to six rats per group.
One-way ANOVA was used to determine significance; only probe sets
that changed significantly with p < 0.001 are shown.

Table 4. Blood: representative genes that changed significantly
(p < 0.001) after HCB treatment were functionally grouped--immune
system. (a)

                                                       Fold change

                                                   HCB low   HCB high
Accession number             Gene name               dose      dose

Granulocytes and
    macrophages
  AA957003          S100 calcium binding protein      4.7       34
                      A8
  L18948            S100 calcium binding protein      4.7       19
                      A9
  L06040            12-lipoxygenase                   1.6        3.6
  U49062            Heat stable antigen CD24         -1.2       -3.0

Mast cells
  U67913            Mast cell protease 10            17         16
  U67911            Mast cell protease 8              3.9        4.6
                      precursor
  X61654            CD63                              1.7        2.0

Pattern
    recognition
    molecule
  AA875213          Peptidoglycan recognition         4.3        7.7
                      molecule

Complement
  AA818025          CD59 protein precursor            1.6        2.6

Cell adhesion
  AF072411          Acid translocase/CD36             2.4        3.6
                      antigen
  AJ009698          Embigin                           1.9        2.0
  D00913            Intercellular adhesion            2.2        1.8
                      molecule 1

Chemokines
  E13732            CC chemokine receptor             1.3        2.4
  U90610            CXC chemokine receptor            2.2        1.1
                      (CXCR4)

Anti-inflammatory
    response
  Al171962          Annexin 1 (p35)                   2.1        4.1

Genes associated
    with T and B
    cells and
    MHCII
    expression
  X76697            CD52/B7 antigen                   1.6        2.1
  X53517            CD37 antigen                     -1.2       -1.8
  Z49761            RT1.Ma                           -1.4       -1.8
  D13555            T-cell receptor CD3, subunit     -1.6       -2.0
                      zeta
  X53430            CD3d antigen (T3 delta)          -1.5       -2.0
  X53054            RT1.D beta chain                 -1.4       -2.1
  X13044            MHC-associated invariant         -1.5       -2.3
                      chain [gamma]
  M15562            MHC class II RT1.u-D-alpha       -1.3       -2.5
                      chain
  U39609            Anti-nerve growth factor 30      -1.5       -2.7
                      antibody light-chain,
                      variable and constant
                      regions

(a) Table contains GenBank accession numbers (http://www.ncbi.nih.gov/
entrez/query.fcgi?db=nucleotide) of cDNA fragments present on
Affymetrix RG U34A gene chips, gene name, and average fold change in
expression of both low dose and high dose versus control. Fold changes
are calculated with data from five to six rats per group. One-way
ANOVA was used to determine significance; only probe sets that changed
significantly with p < 0.001 are shown.

Table 5. Liver: representative genes that changed significantly
(p < 0.001) after HCB treatment--immune system. (a)

                                                       Fold change

                                                    HCB low   HCB high
Accession number             Gene name               dose       dose

Granulocytes and
    macrophages
  AA946503         Lipocalin 2                        4.3     210
  L18948           S100 calcium binding protein       3.4      28
                     A9 (MRP-14)
  AA957003         S100 calcium binding protein       1.1        8.5
                     A8 (MRP-8)
  X76489           CD9 for cell surface               1.4        3.6
                     glycoprotein
  Al104781         Arachidonate 5-lipoxygenase       -1.1        2.3
                     activating protein
  AA893191         ESTs: phosphatidic acid            1.2        2.0
                     phosphatase type 2c
  M55532           Carbohydrate binding receptor      1.1        1.6
                     (Kupffer cell receptor)
  S79263           Interleukin-3 receptor beta        1.7        1.3
                     subunit (colony stimulating
                     factor 2 receptor beta 1,
                     low affinity
                    (granulocyte-macrophage)

Mast cell
  U67911           Mast cell protease 8 precursor     2.2        2.8

Complement
  Z50051           Complement component 4 binding     1.3        2.3
                     protein, alpha

Cell adhesion
  D00913           Intercellular adhesion             1.2        2.3
                     molecule 1

Chemokine
  D11445           Gro                                1.6       11.5

Cytokines
  AA892553         STAT-1                             1.1        3.3
  U77777           Interleukin 18                     1.3        1.9
  L25785           Transforming growth factor        -1.5       -1.5
                     beta stimulated clone 22

Genes associated
    with T and B
    cells and
    MHCII
    expression
  L22654           Antiacetylcholine receptor        -1.0        8.8
                     antibody, rearranged immuno-
                     globulin gamma-2a chain,
                     VDJC region
  U39609           Anti-NGF30 antibody light-
                     chain mRNA, variable and         1.9        8.7
                     constant regions
  X68782           Immunoglobulin heavy chain         1.4        4.6
                     VDJ-region CH1-CH2
  X53054           RT1.D beta chain                   1.5        2.0

(a) Table contains GenBank accession numbers (http://www.ncbi.nih.gov/
entrez/query.fcgi?db=nucleotide) of cDNA fragments present on
Affymetrix RG U34A gene chips, gene names, and average fold change in
expression of both low dose and high dose versus control. Fold changes
are calculated with data from five to six rats per group. One-way
ANOVA was used to determine significance: only probe sets that changed
significantly with p < 0.001 are shown.

Table 6. Kidney: representative genes that changed significantly
(p < 0.001) after HCB treatment--immune system. (a)

                                                       Fold change

                                                    HCB low   HCB high
Accession number             Gene name               dose       dose

Granulocytes and
    macrophages
  L18948           S100 calcium binding protein       1.2       9.6
                     A9
  AA957003         S100 calcium binding protein      -1.7       3.8
                     A8
  M32062           Fc gamma receptor                  1.2       2.7
  U10894           Allograft inflammatory factor     -1.1       2.5
  AA946503         Lipocalin 2                        1.1       2.0
  U49062           Heat stable antigen CD24           1.1       1.8

Complement
  X71127           Complement protein C1q beta        1.3       4.0
                     chain
  D88250           Complement component 1,            1.1       2.9
                     subcomponent

Cell adhesion
  M84488           Vascular cell adhesion             1.0       3.0
                     molecule 1
  D00913           Intercellular adhesion             1.0       2.0
                     molecule 1
  U82612           Fibronectin 1                      1.0       1.6
  Al176461         Selectin, endothelial cell,        1.3      -1.5
                     ligand

Chemokine
  U17035           Chemokine (CXC motif)             -1.1       1.8
                     ligand 10

Cytokines and
    cytokine-
    associated
    genes
  M63122           Tumor necrosis factor receptor     1.1       1.9
  U48596           MAPK kinase kinase 1               1.2       1.9
  M92340           Interleukin 6 signal               1.0       1.5
                     transducer
  S79676           Interleukin 1 beta converting     -1.2       1.4
                     enzyme
  U73142           p38 MAPK                          -1.1       1.3

Genes associated
    with T and B
    cells and
    MHCII
    expression
  L22654           Anti-acetylcholine receptor        2.6       5.3
                     antibody rearranged immuno-
                     globulin gamma-2a chain,
                     VDJC region
  AJ223184         DORA protein (immunoglobulin      -1.4       2.6
                     superfamily member 6)
  U75411           Antiidiotype Ig M light chain     -1.0       2.0
  X13016           MRC OX-45 surface antigen          1.1       1.6
  AF029240         MHC class Ib RT1.S3                1.0       1.4
  S59893           La=autoantigen SS-B/La             1.0       1.4
  X56596           MHC class II antigen RT1.B-1       1.3       1.3
                     beta chain
  X53054           RT1.D beta chain                   1.5       1.2
  M15562           MHC class II RT1.u-D-alpha        -1.3      -2.5
                     chain

(a) Table contains GenBank accession numbers (http://www.ncbi.nih.gov/
entrez/query.fcgi?db=nucleotide) of cDNA fragments present on
Affymetrix RG U34A gene chips, gene name, and average fold change in
expression of both low dose and high dose versus control. Fold changes
are calculated with data from five to six rats per group. One-way
ANOVA was used to determine significance; only probe sets that changed
significantly with p < 0.001 are shown.

Table 7. Representative genes that changed significantly (p < 0.001)
after HCB treatment were functionally grouped: APR and oxidative
stress. (a)

                                                      Fold change

                                                   HCB low   HCB high
Accession number             Gene name              dose       dose

Spleen
  U24441           Matrix metalloproteinase-9        1.1       7.4
                     (gelatinase B)
  M58040           Transferrin receptor             -1.1       7.1
  Al233261         Glutamate-cysteine ligase         1.2       5.0
  K01933           Haptoglobin                       1.3       4.2
  U06099           Thiol-specific antioxidant        1.2       3.0
                     (peroxiredoxin 2)
  D38380           Transferrin                       1.0       2.1
  M11794           Metallothionein-1 and-2           1.1       2.0
  L33869           Ceruloplasmin                     1.0       1.9
  AA944397         Heat shock protein 86             1.2       1.8
  X07365           Glutathione peroxidase            1.4       1.7
  Y00497           Manganese-containing             -1.0       1.6
                     superoxide dismutase
  Al170613         Heat shock 10 kD protein 1        1.1       1.3
  M21060           Copper-zinc containing            1.0       1.3
                     superoxide dismutase
  D00680           Plasma glutathione peroxidase    -1.2      -3.5
                     precursor

MLN
  D00680           Plasma glutathione peroxidase     2.0       4.3
                     precursor
  Y00497           Manganese-containing              1.8       2.6
                     superoxide dismutase
  AA817854         Ceruloplasmin                     1.0       2.2
  S72594           Tissue inhibitor of               1.5       2.0
                     metalloproteinase-2

Blood
  AA926149         Catalase                          1.7       2.8
  Al236795         ESTs, similar to mouse HSP 84    -1.1      -1.6
  M11942           70 kd heat-shock-like protein    -1.1      -1.9

Liver
  L32132           Lipopolysaccharide binding        1.7       8.3
                     protein
  Al169327         Tissue inhibitor of               1.0       6.9
                     metalloproteinase-1
  V01216           Orosomucoid 1                     3.1       6.1
  J02722           Heme oxygenase                    1.8       5.2
  L33869           Ceruloplasmin                     1.4       2.0
  Y00497           Manganese-containing              1.4       1.6
                     superoxide dismutase
  X12367           Glutathione peroxidase I         -1.3      -1.8

Kidney
  L33869           Ceruloplasmin                     1.3       4.2
  D38380           Transferrin                       1.3       2.7
  X68041           Epididymal secretary              1.4      -1.6
                     superoxide dismutase

(a) Table contains GenBank accession numbers (http://www.ncbi.nih.gov/
entrez/query.fcgi?db=nucleotide) of cDNA fragments present on
Affymetrix RG U34A gene chips, gene name, and average fold change in
expression of both low dose and high dose versus control. Fold changes
are calculated with data from five to six rats per group. One-way
ANOVA was used to determine significance; only probe sets that changed
significantly with p < 0.001 are shown.

Table 8. Representative genes that changed significantly (p < 0.001)
after HCB treatment were functionally grouped: enzymes involved in
drug metabolism, porphyria, and estrogen metabolism. (a)

                                                       Fold change

                                                    HCB low   HCB high
Accession number             Gene name               dose       dose

Spleen
  AA800745         Aminolevulinate, delta-,          -1.4       10.7
                     dehydratase
  X06827           Porphobilinogen deaminase          1.2        8.9
                     (hydroxymethylbilane
                     synthase)
  Y00350           Uroporphyrinogen decarboxylase    -1.0        4.0
  D50564           Mercaptopyruvate                   1.1        2.8
                     sulfurtransferase
  AA859700         ESTs, highly similar to ppox,     -1.1        2.5
                     mouse protoporphyrinogen
                     oxidase
  Al176856         Cytochrome P450 1b1                1.5        1.9
  M10068           NADPH-cytochrome P-450            -1.0       -1.3
                     oxidoreductase
  X04229           Glutathione S transferase Y(b)    -1.1       -1.5
                     subunit
  S82820           Glutathione S-transferase Yc2     -1.0       -1.7
                     subunit

MLN
  U36992           Cytochrome P450 7b1                1.4        2.6

Blood
  A1228110         UDP-glucuronosyltransferase 8      1.8        3.8
  D50564           Mercaptopyruvate                   1.7        2.4
                     sulfurtransferase

Liver
  E00778           Cytochrome P450, family 1,         65       125
                     subfamily a, polypeptide 1
  J02852           Cytochrome P450 IIA3               6.4       46
  S76489           Estrogen sulfotransferase          20        43
                     isoform 3
  K00996           Cytochrome P450e                   11        13
                     (phenobarbital-induced)
  M13646           Pregnenolone 16-alpha-             3.2       12
                     carbonitrile-inducible
                     cytochrome P450
  L24207           Testosterone 6-beta-               5.9        6.9
                     hydroxylase (CYP3A1)
  J02722           Heme oxygenase                     1.8        5.2
  E01184           P-450 MC substituted the C         3.0        5.2
                     terminal region cytochrome
                     containing HR2 region for
                     the same region of CYPd
  D86297           Aminolevulinate synthase 2,        2.1        4.4
                     delta
  S82820           Glutathione S-transferase Yc2      3.5        3.4
                     subunit
  M26125           Epoxide hydrolase                  2.7        2.8
  M13506           Liver UDP-glucuronosyl-            2.8        2.7
                     transferase, phenobarbital-
                     inducible form
  S72505           Glutathione S-transferase Yc1      1.7        1.6
                     subunit
  J03914           Glutathione S-transferase Yb       1.9        1.8
                     subunit
  X60328           Cytosolic epoxide hydrolase       -1.7       -3.1
  X91234           17-Beta hydroxysteroid            -1.9      -18
                     dehydrogenase type 2

Kidney
  Al176855         Cytochrome P450, subfamily 1B,     1.1        2.9
                     polypeptide 1
  M37828           Cytochrome P450 4a10               1.2        2.7
  L19998           Minoxidil sulfotransferase         1.1        2.3
  M20131           Cytochrome P450 IIE1              -1.4       -1.9

(a) Table contains GenBank accession numbers (http://www.ncbi.nih.gov/
entrez/query.fcgi?db=nucleotide) of cDNA fragments present on
Affymetrix FIG U34A gene chips, gene name, and average told change in
expression of both low dose and high dose versus control. Fold changes
are calculated with data from five to six rats per group. One-way
ANOVA was used to determine significance; only probe sets that changed
significantly with p < 0.001 are shown.

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Janine Ezendam, (1,2,3) Frank Staedtler, (4) Jeroen Pennings, (2) Rob J. Vandebtiel, (2) Raymond Pieters, (1) Johannes H. Harleman, (5) and Joseph G. Vosa, (3)

(1) Institute for Risk Assessment Sciences (IRAS), Immunotoxicology, Utrecht University, Utrecht, the Netherlands; (2) Laboratory for Toxicology, Pathology and Genetics, National Institute for Public Health and the Environment, Bilthoven, the Netherlands; (3) Faculty of Veterinary Medicine, Department of Pathobiology, Utrecht University, Utrecht, the Netherlands; (4) Biomarker Development and (5) Preclinical Safety, Novartis Pharma AG, Basel, Switzerland

Address correspondence to J. Ezendam, IRAS, Yalelaan 2, 3584 CM, Utrecht, the Netherlands. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

We thank S. Bongiovanni, M. Goetschy, S. Laurent, and N. Hartmann (Novartis, Basel, Switzerland) for performing the DNA microarray experiments. We thank M. de Baets (Department of Neurology,, Academic Hospital of Maastricht, Maastricht, the Netherlands) for performing the radioimmunoassay to determine antiacetylcholine receptor antibodies, and we thank B. Baumann (RIVM, Bilthoven, the Netherlands) for measuring the dioxin-like contamination of HCB.

The authors declare they have no competing financial interests.

Received 13 November 2003; accepted 7 April 2004.