An uncommon pattern of polyneuropathy induced by lifetime exposures to drift containing organophosphate pesticides.

The natural history of chronic peripheral polyneuropathy following lifetime low-level organophosphate (OP) exposure was investigated. A pilot study (1984-1987) conducted in rural communities in Israel detected subtle reversible in-season changes in nerve conduction patterns of 17 field workers out of 214 residents exposed to seasonal drift containing OP's. We examined 60 individuals (males: 50/60; 83.3%) from the original cohort still residing (more than 40 years) in the same communities. Exposure assessment was based on reports by Israeli institutions and the Bureau of Statistics. Information on personal status, work experience, exposures and symptoms was collected by questionnaires. The nervous system was systematically studied, evaluating cortical upper motor neurons, corticospinal tracts, lower motor neurons and peripheral nerves. Electrophysiological studies included conduction velocities, amplitudes and distal latencies of sensory and motor median, ulnar, tibial and sural nerves; F-waves for proximal nerve functions; thermal and pain thresholds for small thinly-myelinated and non-myelinated fibers; transcranial magnetic stimulation for large fibers. Clinical and electrophysiological features of Carpal Tunnel Syndrome were found in 18% of the subjects, atypically in males only. Fingertips' tingling correlated with both axonal and myelin-dependent parameters (lower wave amplitudes and prolonged latency periods, respectively) in the sensory median nerves bilaterally. OP exposure significantly correlated to prolonged distal latency in the right median sensory nerve (r=0.29; p=0.052; n=45) and lower wave amplitude in the right sural nerve (p=0.031). These findings attest to subtle, predominantly sensory peripheral polyneuropathy following lifetime low-level exposures to drifts containing OP.

Novel peptides as potential treatment of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a loss of immunologic tolerance, production of auto-antibodies, and inflammatory damage in multiple organs. We have tested the effect of anti-inflammatory peptide, a H2A histone fragment, termed IIIM1, on MRL/lpr mice, animal model of SLE. Oral administration of IIIM1 at early stage of disease caused reduction in proteinuria and serum anti-dsDNA antibodies. Starting the treatment at advanced stage of disease resulted in prolonged animal survival, decreased lymphadenosis and reduced levels of pathogenic or abnormal double negative CD4(-)CD8(-) cells and B220(+) cells in lymph nodes and spleen. We discovered that IIIM1 induces the production of an additional peptide, a fragment of alpha-1-antitrypsin, termed UBE. A relatively low dose (1 µg/kg) of UBE reduced proteinuria and hematuria in MRL/lpr mice. The beneficial effect of the peptide was corroborated by histological examination. Furthermore a significant reduction in serum IL17, IL12 and anti dsDNA antibodies was observed in the UBE-treated mice. Isolated CD4 cells incubated with the peptide showed a similar cytokine profile. Decreased levels of double negative CD4(-)CD8(-) and B220(+) cells were determined in lymph organs of UBE-treated animals. The beneficial effects of both UBE and IIIM1 suggest these peptides as potential drugs for SLE.

Effects of iodine on inducible nitric oxide synthase and cyclooxygenase-2 expression in sulfur mustard-induced skin.

In a previous study we demonstrated the protective effect of topical iodine as postexposure treatment for sulfur mustard (SM) application. The iodine treatment results in significantly reduced inflammation and necrosis and increased epidermal hyperplasia. The expression and localization of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in paraffin-embedded skin samples from that study were evaluated in the present investigation. We compared the immunoreactivity of iNOS and COX-2 using five samples from each of the following four test sites: untreated control sites, SM-exposed sites, sites treated with iodine mixture 15 min after SM exposure, and sites treated with iodine 30 min after SM exposure. All animals were killed 2 days after irritant exposure. iNOS immunoreactivity was present only in skin sites exposed to SM without iodine treatment. The ulcerated skin was covered with a relatively thick band of exudate composed of iNOS-immunostained polymorphonuclear cells and macrophages. In untreated skin, COX-2 immunostaining was limited to the thin suprabasal epidermal layer. In SM-exposed skin, induction of COX-2 was noted in inflammatory cells located close to the site of epidermal injury. In skin sites treated with iodine 15 or 30 min after SM exposure, the regenerating hyperplastic epithelium showed moderate cytoplasmic staining localized to the epithelium overlying the basal layer. This pattern of staining was also present in the nearby dermal fibroblasts. Thus, in contrast to the skin samples exposed to SM without iodine treatment, the epidermal layer expressing immunohistochemical positivity for COX-2 was thicker and corresponded to the epidermal hyperplasia noted in samples treated with iodine. It is well documented that prostaglandins (PGs) promote epidermal proliferation, thereby contributing to the repair of injured skin. That the induction of the COX-2 shown in our study may also play a role in the healing process is indicated by the present evidence. The results suggest that nitric oxide radicals (NO*) are involved in mediating the damage induced by the SM and that iodine-related reduction in acute epidermal inflammation is associated with reduced iNOS expression.

Topical iodine preparation as therapy against sulfur mustard-induced skin lesions.

Sulfur mustard (SM) is a powerful vesicant employed as an agent of chemical warfare. This study demonstrates the therapeutic effect of a novel topical iodine preparation as a postexposure treatment against SM-induced lesions in the fur-covered guinea-pig skin model. Iodine treatment 15 min after SM exposure resulted in statistically significant reductions of 48, 50, and 55% in dermal acute inflammation, hemorrhage, and necrosis, respectively, whereas, the epidermal healing markers, hyperkerathosis and acanthosis, were significantly elevated by 72 and 67%, respectively, 2 days after treatment. At the interval of 30 min between SM exposure and iodine treatment, there was a significant degree of healing or recovery, albeit to a lesser extent than that observed in the shorter interval. Although the epidermal healing markers were not elevated, the parameters indicative of active tissue damage, such as subepidermal microblisters, epidermal ulceration, dermal acute inflammation, hemorrhage, and necrosis, were significantly reduced by 35, 67, 43, 39, and 45%, respectively. At the 45-min interval between exposure and treatment, there was also a certain degree of healing or recovery expressed as significant reductions in dermal subacute inflammation, subepidermal microblister formation, and epidermal ulceration, whereas, acanthosis was statistically elevated, indicating an increased healing potential. At the 60-min interval, iodine was less efficacious; nevertheless, a significant reduction in the incidence of subepidermal microblisters and an expansion of the acanthotic area were observed. Gross ulceration was significantly decreased at intervals of 15 and 30 min between exposure and treatment. The local anesthetic, lidocaine, did not alter the therapeutic effect of iodine. SM was not affected chemically by iodine as measured by gas chromatography-mass spectrometry (GC-MS) analysis. These findings suggest that the iodine preparation functions as an antidote against skin lesions induced by SM.

Protective effect of povidone iodine ointment against skin lesions induced by chemical and thermal stimuli.

Mustard gas (sulfur mustard, HD) is a powerful vesicant employed as a chemical weapon. The present study demonstrates the effect of povidone iodine (PI) ointment against skin toxicity caused by HD. Gross and histopathological examinations showed that application of PI 20 min or less following exposure to the vesicant resulted in marked skin protection. The shorter, interval between exposure and treatment, the better was the protection achieved. Povidone iodine was also effective against other mustards, such as carboxybutylchloroethyl sulfide (CBCS) and mechlorethamine. The fact that PI protected the skin against agents that cannot be oxidized, such as iodoacetic acid, divinylsulfone and cantharidine, indicated that the antidotal effect of PI was unrelated to oxidation of the nitrogen and sulfur atoms of the mustards. Furthermore, NMR spectroscopy of CBCS treated with iodine did not show oxidation of the sulfur atom. Clinical experience with patients after accidential heat burns (mostly of grade I) has shown that topical application of PI ointment immediately after the stimulus significantly reduced, and often prevented, skin lesions. Apart from being a safe and widely used disinfectant, PI ointment is recommended as an efficient protective agent against skin toxicity caused by hazardous chemicals and by heat stimuli.

Release of amino acids, fluorescamine-reactive substances and substance P from the epidermis of the living animal.

The present study demonstrates a procedure for extraction and determination of stratum corneum amines in the living animal. A nonleaky well, containing 10 mM Tris-HCl buffer, pH 7.0, was constructed on the shaved backs of anesthetized animals. It was found that Ser, Ala, Gly and Pro are mainly released from the stratum corneum of 4-month-old guinea pigs, and in 2-month-old rats, Gly, Ser and Arg show the highest degree of release. Much lower amino acid concentrations were observed in 20-month-old rats. This was also reflected by the high levels of fluorescamine-reactive substances released from young rat skin as compared to the old animals. The release of the neuropeptide substance P into the aqueous medium was increased 3.2 times upon heat stimulus as compared to control skin. Amines and other compounds released from the skin may serve as markers for skin aging or for certain skin disorders, leading to a new approach for their treatments.

Skin surface proteolytic activity. Partial characterization and identification.

Skin surface proteolytic activity in the living animal was determined by a sensitive, non-invasive methodology developed in our laboratory. A non-leaky well was constructed on the shaved back of an anesthetized guinea pig. The well contained the reaction mixture including the substrate 125I-S-carboxymethylated insulin B-chain (ICMI). The proteolytic activity was shown to be time-dependent. The activity was strongly inhibited by pepstatin A, indicating the involvement of aspartic proteinase(s) such as cathepsin D and/or E. Pretreatment of the skin with propylene glycol blocked the proteolytic activity. The present study demonstrates the presence of proteolytic activity located on skin surface using a unique, non-invasive method for in situ proteinase determination in the living animal.

Protective effect of povidone-iodine ointment against skin lesions induced by sulphur and nitrogen mustards and by non-mustard vesicants.

Mustard gas (sulphur mustard, SM) is a powerful vesicant employed as a chemical weapon. The present study demonstrates the effect of povidone iodine (PI) ointment against skin toxicity caused by SM. Gross and histopathological examinations showed that application of PI up to 20 min following exposure to the vesicant resulted in marked skin protection. The shorter the interval between exposure and treatment the better was the protection achieved. PI was also effective against other mustards such as carboxybutyl chloroethyl sulphide (CBCS) and mechlorethamine. The fact that PI protected the skin against agents which cannot be oxidized such as iodoacetic acid, divinylsulphone and cantharidine showed that the antidotal effect of PI was unrelated to oxidation of the nitrogen and sulphur atoms of the mustards. PI ointment is proposed as an efficient protective agent against skin toxicity caused by mustards and other alkylators.

Noninvasive procedure for in situ determination of skin surface aspartic proteinase activity in animals; implications for human skin.

The present study demonstrated a noninvasive procedure for in situ determination of stratum corneum aspartic proteinase in the living animal. A non-leaky well, containing [125I]S-carboxymethylated insulin B-chain (ICMI) as a substrate, was constructed on the shaved back of anesthetized guinea pigs and rats. The enzymatic activity was determined by measuring the radiolabeled trichloroacetic acid soluble material. We demonstrated pepstatin-sensitive proteinase activity bound to the skin surface indicating the involvement of aspartic proteinase(s) such as cathepsin D and/or E. Aged rats had about six fold lower activity than young animals. The proteinase activity was inhibited by the alkylating agent mechlorethamine and by the cosmetic propylene glycol. A similar procedure was carried out with intact human skin pieces obtained during plastic surgery. The activity was inhibited by antihuman cathepsin D antibodies. Cathepsin D was immunohistochemically localized in the corneal and granular layers of the epidermis. Skin surface aspartic proteinase/cathepsin D activity may serve as a marker for skin aging or for certain skin disorders leading to a new approach to their medical treatments.

In vivo and in vitro toxicity of newly synthesized monofunctional sulfur mustard derivatives.

The toxicity of two new monofunctional sulfur mustard derivatives was tested. The compound (4-carboxybutyl 2-chloroethyl sulfide, CBCS; 10-carboxydecyl 2-chloroethyl sulfide, CDCS) possess the 2-chloroethyl sulfide moiety present in mustard gas. Exposure of guinea pig skin to CBCS resulted in a dose-related ulcerative effect. CDCS exhibited similar pathological effects. Dimethylsulfoxide (DMSO) exacerbated CBCS toxicity. Regeneration and healing were prominent six days after application. Concentration-related effects were found in in vitro systems, using human SH-SY5Y neuroblastoma cells for acute toxicity and Y79 retinoblastoma cells for colony forming assay. CBCS or derivatives may serve as models compounds for investigating the mechanism of action of alkylating agents.

Possible pitfalls in rat extended dermal toxicity testing: an hepatic-ocular syndrome.

During a sub-chronic dermal toxicity study, hepatic and ocular toxicity were detected unrelated to the compound being tested. The changes in the liver were characterized by hepatic centrilobular degeneration and fibrosis due to chronic passive congestion. Clinical chemistry results confirmed the active hepatic damage and cholestasis. The ocular changes were limited to the retina where diffuse retinal atrophy was seen originating adjacent to the optic nerve and progressing centrifugally. It is suggested that as a consequence of the bandaging technique, a state of passive congestion and cholestasis resulted. The decrease in flow of bile acids to the intestine caused disturbances in vitamin E absorption. The deficiency of the antioxidant vitamin was responsible for the retinal changes seen.

Toxicology of mustard gas.

The devastating effects of mustard gas were first observed in World War I. The advent of the Gulf War fueled renewed fears of further use of toxic gases in battle, with the possible exposure of large civilian populations--while understanding of the mechanism of action of the alkylating sulfur mustards was still quite restricted. In this article Uri Wormser discusses the structure--activity studies that are available, and the limited pharmacological measures that can be taken to protect against mustard gas attack. In addition to systemically administered sulfhydryl agents, new percutaneous preparations are being developed in the author's laboratory which offer better protection than is possible with simple adsorbant powders.

Hepatic and renal metallothionein induction in the rat and mouse after treatment with furosemide.

Induction of hepatic and and renal metallothionein by furosemide was studied in the rat and mouse. Treatment of mice with 200 and 300 mg/kg furosemide elevated hepatic metallothionein by 117% and 366%, while renal metallothionein was induced by 29% and 380%, respectively. In the rat the drug was less potent i.e. liver metallothionein was increased by 167% and 217% following injection of 300 and 400 mg/kg furosemide, respectively, whereas kidney was not significantly changed by this treatment. The mouse hepatic and renal metallothionein was identified as zinc-containing thionein by Sephadex G-75 gel filtration (Ve/Vo = 2.0). In both species maximal induction was observed 24 hours post exposure. However, the mouse hepatic and renal metallothionein content declined after additional 24 hours whereas the rat metalloprotein was not reduced even after 72 hours of treatment. It is suggested that alterations in metal homeostasis may be responsible for hepatic and renal metallothionein induction caused by furosemide.

Protective effect of O-phenanthroline against mechlorethamine toxicity in the rat liver slice system and in the guinea pig skin.

The effect of O-phenanthroline (OP) on mechlorethamine hydrochloride (HN2) toxicity was studied in in vitro and in vivo experiments. Incubation of HN2 with the in vitro rat liver slice system resulted in leakage of alanine aminotransferase (ALT) in a time-dependent manner. Exposure of the slices to HN2 for 4 h caused 79.2% ALT leakage. In the presence of OP enzyme leakage was reduced to 28.7%. OP-induced protection was shown to be dose dependent. Other metal chelators such as dithiothreitol (DTT) and EDTA (ethylenediaminetetraacetic acid) had a weak effect on HN2 cytotoxicity. The protective activity of OP was also demonstrated in in vivo skin toxicity studies in the guinea pig. The ulcerative effect of topically applied HN2 was inhibited by OP even when applied 10 min following the alkylator. Histology of NH2-treated skin showed epidermal ulceration associated with a covering layer of encrusted exudate. However, only a slight diffuse acanthosis of the epidermal layer was observed when OP was applied for 10 min after the vesicant. It is suggested that OP may be used for the prevention of tissue damage caused by antineoplastic treatment with nitrogen mustard. It might also be employed in military medicine as an antidote to the chemical weapon sulfur mustard.

Proteolytic resistance and biological activity of N-methylated analogs of [pGlu6] substance P6-11.

Five synthetic N-methylated analogs (II-V) of the C-terminal hexapeptide analog of substance P (SP), [pGlu6]SP6-11 (I) were evaluated for their metabolic stability and in vitro spasmogenic activity. The metabolic resistance of the analogs was tested by two SP degrading systems with different specificities, namely, the rat parotid and the hypothalamic slice systems. Their biological activity was assessed in the isolated guinea pig ileum. The analog [pGlu6, N-Me Phe7, N-Me Gly9]SP6-11 (III), had relative potency of 65% in the spasmogenic assay as compared to the parent compound. It was found to be more stable than the parent peptide in the hypothalamus, whereas in the parotid system it was susceptible as the parent peptide. However, the analog [pGlu6, N-Me Leu10]SP6-11 (II) (46% relative potency in the spasmogenic assay) was more stable than the parent peptide in the parotid system but did not show any improved stability in the hypothalamus. Identification of degradation products of the [pGlu6, N-Me Leu10]SP6-11 reflected the differences in the specificities of the two preparations. A significant drop in potency (7%) was observed for [pGlu, N-Me Phe7]SP6-11 (IV). This analog was more stable in the hypothalamic system than in the parotid. Introduction of a double methylation, [pGlu6, N-Me Leu10] SP6-11, contributed toward the stabilization in both degrading systems. Its relative spasmogenic activity was comparable to that of analog IV. In light of the above mentioned findings the implications of the N-methylated analogs with respect to putative CNS activity are discussed.