Generation of quinoneimine intermediates in the bioactivation of 3-(N-phenylamino)alanine (PAA) by human liver microsomes: a potential link between eosinophilia-myalgia syndrome and toxic oil syndrome.

Eosinophilia-myalgia syndrome (EMS) was an intoxication episode that occurred in the US in 1989 and affected 1,500 people. EMS was associated with the ingestion of manufactured L-tryptophan, and 3-(N-phenylamino)alanine (PAA) was identified as one of the contaminants present in the L-tryptophan batches responsible for intoxication. In previous studies (Martínez-Cabot et al., Chem Res. Toxicol., in press), we have shown that the incubation of 3-(N-phenylamino)propane-1,2-diol (PAP), a toxic biomarker of the oil batches that caused Toxic Oil Syndrome in Spain, with human liver microsomes generates a reactive quinoneimine intermediate. The structural similarity between PAA and PAP led Mayeno and co-workers (Mayeno et al. (1995) Chem. Res. Toxicol. 8, 911-916) to hypothesize that both xenobiotics could be linked to a common etiologic agent. We thus set about to study the bioactivation of PAA by human liver microsomes. Under these conditions, PAA is converted to its 4'-hydroxy derivative, an unstable intermediate that is rapidly transformed into the final metabolites 4-aminophenol and formylglycine, which were identified in the incubations by GC/MS using the H2(18)O-labeled medium. We also provide evidence that 4-aminophenol and formylglycine are formed from a quinoneimine intermediate via a pathway similar to that demonstrated for PAP bioactivation. This quinoneimine, in the absence of nucleophiles in the incubation medium, could isomerize to give the corresponding imine, which could undergo hydrolysis to yield the aforementioned final products. These findings establish that EMS and TOS are linked by a common toxic metabolite (4-aminophenol) and that they may be further linked by the concomitant release of potentially hazardous carbonyl species.

A heretofore undisclosed crux of eosinophilia-myalgia syndrome: compromised histamine degradation.

In contrast to early epidemiological evidence offering links between eosinophilia-myalgia syndrome (EMS) and microimpurities of L-tryptophan-containing dietary supplements (LTCDS), this account shows why reliance on a finite impurity from one manufacturer is both unnecessary and insufficient to explain the etiology of EMS. Excessive histamine activity has induced blood eosinophilia and myalgia (Greek: mys, muscle + algos, pain). Termination of the multiple actions of histamine is dependent on particular amine oxidases and histamine-N-methyltransferase. Histamine metabolism is rapid when these degradative reactions are operative. The latent effects of incurred histamine can be potentiated and aggravating when these mechanisms are impaired. Overloads of tryptophan supplements cause - among other relevant side-effects - an increased formation of formate and indolyl metabolites, several of which inhibit the degradation of histamine. Moreover, (non-EMS) subjects with hypothalamic-pituitary- adrenal (HPA) axis dysregulation have also manifested greatly increased sensitivities to incurred tryptophan and histamine. A final common pathway for syndromes characterized by eosinophilia with myalgia is now evident.

A contaminant of L-tryptophan enhances expression of dermal collagen in a murine model of eosinophilia myalgia syndrome.

The eosinophilia-myalgia syndrome was associated with the ingestion of L-tryptophan products containing a number of contaminants, one of which has been identified as 1,1'-ethylidene-bis-(L-tryptophan) (EBT), also known as peak E or peak 97. In earlier studies, we demonstrated that EBT induces inflammation and fibrosis in dermal and subcutaneous tissue of C57BL/6 mice. Others have shown EBT to be a potent stimulus for fibroblast activation and collagen synthesis in vitro, and dermal tissue from EMS patients reveals evidence of enhanced collagen gene expression. In the present study using Northern blot analysis and in situ hybridization, we demonstrate enhanced expression of genes for types I, III, and VI collagen in the dermis and subcutis of C57BL/6 mice treated with EBT for 3-21 days. Increased type I procollagen mRNA was noted on day 6 of EBT treatment and was followed by enhanced expression of type III and VI procollagen mRNA at day 21. L-Tryptophan, free of contaminants associated with the eosinophilia-myalgia syndrome epidemic, increased dermal collagen mRNA to a lesser extent than did EBT. Increased procollagen gene expression was accompanied by evidence of enhanced TGF-beta 1 expression in the dermis and subcutis. This animal model provides additional evidence for EBT as a causal agent of the eosinophilia-myalgia syndrome and should prove useful in the study of the pathogenesis of that syndrome.

EBT, a tryptophan contaminant associated with eosinophilia myalgia syndrome, is incorporated into proteins during translation as an amino acid analog.

The tryptophan dimer 1,1'-ethylidenebis[L-tryptophan] was identified as a contaminant of tryptophan preparations associated with Eosinophilia-Myalgia Syndrome. In this paper, we describe experiments examining the hypothesis that 1,1'-ethylidenebis[L-tryptophan] acts as an amino acid analog replacing L-tryptophan during the synthesis of proteins. We propose further that proteins containing 1,1'-ethylidenebis[L-tryptophan] are rejected in an autoimmune process identified clinically as Eosinophilia-Myalgia Syndrome. Rabbit reticulocyte lysates containing an estimated 1 microM L-tryptophan were used to assay the ability of 1,1'-ethylidenebis[L-tryptophan] to compete with 3H-L-tryptophan for incorporation into proteins translated from BMV RNA. 1,1'-Ethylidenebis[L-tryptophan] in concentrations of 40, 80 and 110 microM reduced lysate 3H-L-tryptophan incorporation to 81%, 76% and 75% of control incorporation obtained in the absence of 1,1'-ethylidenebis[L-tryptophan]. In the presence of 20 microM L-tryptophan, 110 microM 1,1'-ethylidenebis[L-tryptophan] reduced 3H-L-tryptophan incorporation to 56% of control incorporation. In contrast, ethyl-L-tryptophan did not significantly reduce 3H-L-tryptophan incorporation. In the presence of 110 microM 1,1'-ethylidenebis[L-tryptophan] and 20 microM L-tryptophan, 3H-L-leucine incorporation was not significantly reduced compared to incorporation in the absence of 1,1'-ethylidenebis[L-tryptophan], demonstrating that proteins were translated to full length during elongation. These findings suggest that 1,1'-ethylidenebis[L-tryptophan], but not ethyl-L-tryptophan, reduced 3H-L-tryptophan incorporation into proteins by substituting for L-tryptophan rather than by causing premature termination or significant slowing of nascent protein chains.

Enhanced collagen synthesis and transcription by peak E, a contaminant of L-tryptophan preparations associated with the eosinophilia myalgia syndrome epidemic.

The pathogenesis of the eosinophilia myalgia syndrome (EMS) remains unclear. Several abnormal constituents have been found in the L-tryptophan lots responsible for the illness, particularly, 1,1-ethylidenebis[L-tryptophan], also called peak E or EBT, and 3-phenylamino-alanine or peak 5. However, the role of these contaminants in the pathogenesis of EMS and in the development of fibrosis is unknown. We now report that peak E, a dimer of L-tryptophan, is a potent stimulus for human dermal fibroblast DNA and collagen synthesis. Peak E (0.1-1.0 microM) increased DNA synthesis up to four-fold (P = 0.0001) in a dose-dependent manner (r = 0.987). When added to monolayer cultures for 2 to 24 h, peak E (0.5 to 100 microM) caused a progressive, more than threefold increase in alpha 1(I) procollagen mRNA levels and collagenous protein. No increase in procollagen mRNA levels was found after the addition of another major L-tryptophan contaminant, peak 5, or with L-tryptophan itself. Transient transfection with a 2.5-kb alpha 1(I) procollagen promoter-luciferase construct showed that peak E causes a twofold upregulation of promoter activity (P = 0.022). Contraction of collagen gels, consisting of human dermal fibroblasts incorporated into a type I collagen lattice, was enhanced two-fold by exposure to peak E (P = 0.001). We conclude that a major constituent of contaminated batches of L-tryptophan, peak E, is a potent stimulus for fibroblast activation and collagen synthesis. This stimulatory action of peak E may provide a direct mechanism for the development of fibrosis in EMS.

Pharmacogenetic characteristics of the eosinophilia-myalgia syndrome.

This study tested the hypothesis that patterns of xenobiotic metabolism in patients with eosinophilia-myalgia syndrome (EMS) differed from healthy control subjects. We determined the genotypes of 27 EMS patients with EMS and 114 control subjects for the cytochrome P450 CYP2D6 polymorphism. The metabolic phenotypes of patients with EMS for S-mephenytoin hydroxylation (n = 17) and dapsone acetylation (n = 19) were determined and compared with 29 healthy control subjects. The incidence of the CYP2D6 poor metabolizer genotype (mutant/mutant) was 0.185 in patients with EMS and 0.061 in control subjects (Mantel-Haenszel, chi 2 = 7.213, p = 0.007). The mephenytoin S/R ratios were 0.39 +/- 0.23 in patients with EMS versus 0.18 +/- 0.13 in control subjects (p < or = 0.005). There was no difference in dapsone acetylation between the two groups. A pattern of xenobiotic metabolism may play a role in the pathogenesis of EMS, but the precise role that it plays remains unclear.

Immunohistochemical analysis of lymphocytes in postmortem study of the heart from fatal cases of the eosinophilia myalgia syndrome and of the toxic oil syndrome.

Inflammatory lesions of coronary arteries and cardiac neural structures are postmortem histopathologic features of both the eosinophilia-myalgia syndrome (EMS) and the toxic oil syndrome (TOS). The inflammation is primarily lymphocytic. For further definition of the lymphocytes, immunohistochemical analysis was carried out in the hearts of three victims of EMS and four victims of TOS. Many CD45RO+ T cells, OPD4+ helper/inducer T (Th) cells, and CD20+ B cells were observed in these neurovascular lesions, notably in the conduction system and the coronary chemoreceptor. T cells were prominent in EMS around nerves, ganglia, and sometimes around arteries. B cells and Th cells, however, were more prominent in TOS around arteries. The percentage of T cells in EMS (59.6 +/- 2.4%) was significantly higher than in TOS (45.0 +/- 4.2%), whereas that of B cells was significantly higher in TOS (27.7 +/- 4.4%) than in EMS (17.5 +/- 1.3%) (p < 0.01, respectively). There was no significant difference between the syndromes in the percentages of Th cells. Therefore cytotoxic/suppressor T cells are more prominent in EMS than in TOS. These findings suggest that (1) cellular immune mechanisms are involved in cardioneuropathy in victims of both EMS and TOS; (2) cell-mediated cytotoxicity directed against chemoreceptor neural structures and sinus nodal myocytes is prominent in EMS; and (3) some humoral factors may also be involved in the pathogenesis of TOS.

Studies with 1,1'-ethylidenebis(tryptophan), a contaminant associated with L-tryptophan implicated in the eosinophilia-myalgia syndrome.

L-Tryptophan binds to a rat liver nuclear envelope protein, and this binding is saturable, stereospecific, and of high affinity. Utilizing an in vitro assay of [3H]tryptophan binding to rat hepatic nuclear envelopes, we have previously determined that the L-tryptophan obtained from Showa Denko and which was implicated in cases of the eosinophilia-myalgia syndrome (EMS) inhibited [3H]tryptophan binding differently than did control L-tryptophan (not implicated in EMS). Therefore, in this study we investigated whether the addition of 1,1'-ethylidenebis(tryptophan) (EBT), a contaminant or impurity in L-tryptophan implicated in EMS, would have an effect. Our results indicate that EBT alone has little inhibitory binding effect compared with that of control L-tryptophan and that when EBT was added to control L-tryptophan the inhibitory binding effort was similar to that of control L-tryptophan alone. On the other hand, in vitro addition of EBT plus L-tryptophan to nuclei of cultured murine macrophages (WLG5) results in less inhibition of [3H]-tryptophan binding than does addition of L-tryptophan alone. Similar in vitro additions to nuclei of rat brain reveal little effect on binding, as was also the case for hepatic nuclear envelopes. Adding EBT to an in vitro hepatic protein synthesis system and measuring [3H]tryptophan incorporation into acid-precipitable proteins reveal that it competes similarly to that found with equimolar concentrations of unlabeled L-tryptophan. It does not affect [14C]leucine incorporation into proteins. [14C]EBT becomes incorporated in vitro into proteins (acid-precipitable), and this incorporation is diminished in the presence of equimolar concentrations of unlabeled EBT or L-tryptophan. This suggests that EBT or possibly a breakdown product becomes incorporated into proteins. Speculation as to how EBT may affect tissues in experimental animals is presented.

P-31 magnetic resonance spectroscopy of skeletal muscle in the eosinophilia-myalgia syndrome: a preliminary study.

OBJECTIVE: To determine the pathogenesis of muscular symptomatology in the eosinophilia-myalgia syndrome (EMS). METHODS: P31-nuclear magnetic resonance spectroscopy (MRS) was performed on 7 patients with EMS and 10 age matched controls to determine the relative concentrations of phosphocreatine (PCr), inorganic phosphate (Pi), and adenosine triphosphate (ATP) in calf muscle. RESULTS: Patients with EMS had statistically lower ATP/PCr ratios and higher Pi/ATP ratios than controls, indicating a preferential and unique decrease in ATP levels at rest in skeletal muscle. CONCLUSION: Skeletal muscle is metabolically abnormal in patients with EMS. This abnormality may contribute to the myalgia, spasm, and weakness seen in this disorder.

Eosinophilia-myalgia syndrome: findings at MR imaging and proton spectroscopy of the lower leg.

Five magnetic resonance (MR) studies of the lower leg were performed in three patients with eosinophilia-myalgia syndrome (EMS). The 1H spectroscopic and imaging findings were compared with seven examinations of age-matched healthy controls. Standard imaging with proton density-, T1-, and T2-weighted spin-echo (SE) sequences at 1.5 T showed marked atrophy of the calf muscles and slightly increased signal strength of muscle tissue in T2-weighted SE images. The application of frequency selective chemical shift imaging (SENEX) exhibited skin changes similar to those of scleroderma with increased water content and thickened cutis in the water selective images. In one patient the tibialis muscles showed irregular structures, but no fatty degeneration as demonstrated in the fat selective images. Proton signals from volume elements of (20 mm)3 within the soleus and gastrocnemius muscle were recorded by the PRESS localization method. A reduction of the creatine/water and the choline/water ratios was found in the 1H spectra from the EMS patients compared to the controls. Localized 1H spectroscopy exhibited modified distributions of the lipid signals in two EMS patients with slightly elevated signals from unsaturated fatty acids. The transverse relaxation of choline and creatine signals was accelerated in both examinations of one patient compared with the healthy controls.

Fibrogenic growth factors in the eosinophilia-myalgia syndrome and the toxic oil syndrome.

BACKGROUND AND DESIGN: We sought to determine if growth factors of potential pathogenetic significance are deposited in the skin, muscle, and peripheral nerve lesions of eosinophilia-myalgia (EMS) and toxic oil syndrome. Immunohistochemical studies using affinity-purified peroxidase-conjugated antibodies to detect transforming growth factor-beta, platelet-derived growth factorAA and growth factorBB, fibroblast growth factor, epidermal growth factor, and interleukin 4 were performed on formalin-fixed, paraffin-embedded specimens. Seven skin biopsy specimens from EMS, six skin biopsy specimens from toxic oil syndrome, nine muscle biopsy specimens from EMS, and one sural nerve biopsy specimen from EMS were studied. RESULTS: Growth factor staining was noted primarily in the epidermis and periappendageal locations of the dermis. The presence of TGF-beta and platelet-derived growth factorAA in the periappendageal dermis was significantly more prevalent in EMS than toxic oil syndrome (57% vs 0%). Prominent staining of transforming growth factor-beta was also present in the perimysial connective tissue of five (63%) of eight EMS muscle biopsy specimens and one sural nerve biopsy specimen. CONCLUSIONS: These studies implicate transforming growth factor-beta and platelet-derived growth factorAA as potentially important cytokines in EMS and suggest that the pathogenesis of tissue fibrosis in EMS and toxic oil syndrome may be dependent on different growth factors.

Comparison of the pathology of fascia in eosinophilic myalgia syndrome patients and idiopathic eosinophilic fasciitis.

The L-tryptophan eosinophilic myalgia syndrome (EMS) clinically has some similarities with idiopathic eosinophilic fasciitis (EF). In order to study the pathology of both syndromes, we analyzed 21 biopsies of patients with EMS and 8 with idiopathic EF. In both diseases there is dermal and fascial mucin and dermal edema, but this was more common in EMS. EMS is also characterized by dilated lymphatics, dermal and septal sclerosis and macrophage-rich inflammation. Neural inflammation was seen in 4 of the cases with EMS and in none with idiopathic EF. In both syndromes, there are many histopathological similarities. The differences may be due to sampling and to sample size. The nerve lesions of EMS may result from the nature of lymphocyte-macrophage inflammation, or the effect of the eosinophil neurotoxin and may not be a primary event.

Central nervous system involvement in the eosinophilia-myalgia syndrome.

A patient with eosinophilia-myalgia syndrome developed progressive central nervosa system involvement that did not improve despite discontinuation of L-tryptophan therapy. Neurologic impairment was manifested initially by spastic monoparesis, which was improved by treatment with methyl-prednisolone and hydroxyurea. Recurrence of weakness was accompanied by gait ataxia, dysphagia, and complaints of a gradual decline in memory and concentration. Neuropsychological testing identified a broad pattern of cognitive deficits suggestive of a subcortical dementia, and magnetic resonance imaging demonstrated multiple high-signal lesions in the white matter. Cognitive deficits appear to be underrecognized in patients with the eosinophilia-myalgia syndrome. The response of our patient's initial symptoms to corticosteroid therapy suggests a possible role for autoimmune mechanisms in the pathogenesis of central nervous system involvement in the eosinophilia-myalgia syndrome. Neuropsychological evaluation should be performed in patients with cognitive complaints to delineate the full spectrum of central nervous system impairment associated with the eosinophilia-myalgia syndrome.

Tryptophan metabolism via the kynurenine pathway in patients with the eosinophilia-myalgia syndrome.

OBJECTIVE: To investigate the metabolism of L-tryptophan (LT) via the kynurenine pathway in patients with the eosinophilia-myalgia syndrome (EMS). METHODS: Measurement of LT, L-kynurenine, and quinolinic acid in plasma and cerebrospinal fluid (CSF) from subjects with EMS, from asymptomatic users of LT, and from normal subjects. RESULTS: Plasma LT concentrations were lower in untreated EMS patients (n = 5) than in corticosteroid-treated EMS patients (n = 5; P less than 0.05) and in asymptomatic users of LT (n = 5; P less than 0.05). Untreated EMS patients, who had discontinued LT weeks to months prior to study, had significantly higher plasma levels of L-kynurenine and quinolinic acid than did corticosteroid-treated EMS patients (P less than 0.05), normal subjects (P less than 0.02), and asymptomatic users of LT (P less than 0.05). EMS patients also had significantly elevated levels of L-kynurenine (P less than 0.05) and quinolinic acid (P less than 0.001) in CSF compared with normal subjects. After a 1-gm oral dose of LT, untreated EMS patients (n = 4) showed lower peak levels of LT and accentuated synthesis of L-kynurenine and quinolinic acid, compared with these values in corticosteroid-treated EMS patients (n = 2), who responded like normal subjects (n = 5). CONCLUSION: These data demonstrate that during the active phase of EMS, LT metabolism via the kynurenine pathway was accentuated, probably secondary to induction of the enzyme indoleamine-2,3-dioxygenase. Ingestion of large amounts of LT (median daily dose 1.5 gm) resulted in high concentrations of kynurenine-pathway metabolites in blood and extrahepatic tissues, which was accentuated in EMS patients and which may have played a significant role in the pathogenesis of the disease.

Alterations in tryptophan metabolism in the toxic oil syndrome and in the eosinophilia-myalgia syndrome.

The eosinophilia-myalgia syndrome (EMS) was associated with ingestion of L-tryptophan containing products and was accompanied by altered metabolism of L-tryptophan during the active phase. Many patients with EMS exhibited clinical and histopathological features similar to another epidemic, the toxic oil syndrome (TOS), associated with ingestion of adulterated rapeseed oil. We hypothesized that patients with TOS, like patients with EMS, may have had altered metabolism of L-tryptophan during the acute phase of the illness. Therefore, we quantitated the tryptophan metabolites, L-kynurenine and quinolinic acid, and we measured neopterin, a marker of interferon-gamma (IFN-gamma), in blood obtained during the acute phase of each syndrome. Patients with TOS or EMS had significantly higher L-kynurenine and quinolinic acid than healthy control subjects or rheumatic disease control subjects. Neopterin was also elevated in patients with untreated TOS and EMS, and correlated strongly with L-kynurenine and quinolinic acid. Our data suggest that indoleamine-2,3-dioxygenase (IDO), the rate limiting enzyme of the kynurenine pathway of L-tryptophan metabolism, was activated in both syndromes by cytokines including IFN-gamma, and that perhaps products of tryptophan metabolism played a role in the pathogenesis of EMS and TOS.

[Eosinophilia-myalgia: new syndrome]. / Eozynofilia-mialgia: nowy zespól chorobowy.

The eosinophilia-myalgia syndrome is a newly recognized illness that has been associated with the consumption of tryptophan products. We describe the clinical and histopathological findings and the results of biochemical analyses of tryptophan metabolism in patients with this syndrome and the toxic-oil syndrome which took place in 1981 in Spain. Symptoms and laboratory findings are similar. Chronic phase of EMS is characterised by long-term disability, sclerodermatous skin thickening, sensorimotor polyneuropathy and severe episodic myalgias. The development of the syndrome may result from a confluence of several factors including the ingestion of tryptophan, exposure to agents that activate indoleamine-2,3-dioxygenase, and possibly, impaired function of the hypothalamic-pituitary-adrenal axis.