Lygia T. Budnik

Inhibitory effects of TNFα on mouse tumor Leydig cells: possible role of ceramide in the mechanism of action

TNF alpha is reported to inhibit steroidogenesis in mouse Leydig cells. In primary cells this inhibition resulted mainly from a reduced expression of Cyp-17 gene. Mouse tumor Leydig cells, MA-10, being free of macrophages and lacking Cyp-17, appear to be an excellent model to investigate those effects of TNF alpha which are independent of either macrophages or Cyp-17. We report here that TNF alpha receptors are expressed in this cell line. Treatment of the cells with TNF alpha had no effect on basal progesterone production. In contrast, LH-, 8Br-cAMP and forskolin-stimulated progesterone production was inhibited by TNF alpha. Neither enzymes involved in the conversion of cholesterol to pregnenolone nor hormone-induced hydrolysis of [14C] cholesterol-ester were affected by TNF alpha. The hormone-induced expression of StAR protein was diminished in mitochondrial fractions from TNF alpha-treated cells. Also cell permeable ceramides markedly inhibited StAR protein levels. We show further that TNF alpha was able to induce [14C]-ceramide accumulation in MA-10 cells and suggest that this sphingolipid may be considered as a transmitter of TNF alpha signals to the StAR protein.

Lysophosphatidic acid and its role in reproduction

Abstract Lysophosphatidic acid (LPA) belongs to a new family of lipid mediators that are endogenous growth factors and that elicit diverse biological effects, usually via the activation of G protein-coupled receptors. LPA can be generated after cell activation through the hydrolysis of preexisting phospholipids in the membranes of stimulated cells. A dramatic elevation of LPA levels was found in serum of patients suffering from ovarian carcinoma. Because these high LPA amounts can be detected as early as stage I of the disease, LPA has been introduced as a new marker for ovarian cancer. Progression of the malignancy is correlated with a differential expression of various LPA receptor subtypes. The presence of LPA in the follicular fluid of healthy individuals implicates that this biological mediator may be relevant to normal ovarian physiology. LPA induces proliferation and mitogenic signaling of prostate cancer cells, and a novel LPA receptor isoform has been recognized in healthy prostate tissues. This evidence indicates multiple roles for LPA in both male and female reproductive physiology and pathology. In this review, we summarize the literature on LPA generation, the way it is degraded, and the mechanisms by which signals are transduced by various LPA receptors in reproductive tissues, and we discuss possible future research directions in these areas.

Differences in the carcinogenic evaluation of glyphosate between the International Agency for Research on Cancer (IARC) and the European Food Safety Authority (EFSA)

The International Agency for Research on Cancer (IARC) Monographs Programme identifies chemicals, drugs, mixtures, occupational exposures, lifestyles and personal habits, and physical and biological agents that cause cancer in humans and has evaluated about 1000 agents since 1971. Monographs are written by ad hoc Working Groups (WGs) of international scientific experts over a period of about 12 months ending in an eight-day meeting. The WG evaluates all of the publicly available scientific information on each substance and, through a transparent and rigorous process,1 decides on the degree to which the scientific evidence supports that substances potential to cause or not cause cancer in humans. For Monograph 112,2 17 expert scientists evaluated the carcinogenic hazard for four insecticides and the herbicide glyphosate.3 The WG concluded that the data for glyphosate meet the criteria for classification as a probable human carcinogen . The European Food Safety Authority (EFSA) is the primary agency of the European Union for risk assessments regarding food safety. In October 2015, EFSA reported4 on their evaluation of the Renewal Assessment Report5 (RAR) for glyphosate that was prepared by the Rapporteur Member State, the German Federal Institute for Risk Assessment (BfR). EFSA concluded that ‘glyphosate is unlikely to pose a carcinogenic hazard to humans and the evidence does not support classification with regard to its carcinogenic potential’. Addendum 1 (the BfR Addendum) of the RAR5 discusses the scientific rationale for differing from the IARC WG conclusion. Serious flaws in the scientific evaluation in the RAR incorrectly characterise the potential for a carcinogenic hazard from exposure to glyphosate. Since the RAR is the basis for the European Food Safety Agency (EFSA) conclusion,4 it is critical that these shortcomings are corrected. EFSA concluded ‘that there is very limited evidence for an association between glyphosate-based formulations …

High frequency of fumigants and other toxic gases in imported freight containers – an underestimated occupational and community health risk

Residues of pesticide fumigants and toxic industrial chemicals in freight containers represent a health hazard to employees and consumers, especially since freight containers are sealed for transport and distributed widely throughout the importing countries before being opened for unloading. We investigated 2113 freight containers arriving at the second largest container terminal in Europe, Hamburg, Germany, over a 10-week period in 2006. The countries of origin, type of contents and the pesticide fumigation history declared on labels attached to the container were recorded. We determined that 1478 (70%) containers were contaminated with toxic chemicals above chronic reference exposure levels; 761 (36%) even exceeded the higher acute reference exposure level thresholds. Benzene and/or formaldehyde contamination was 4-times greater than for fumigants. Our findings indicate a health risk for dockworkers, container unloaders and even end-consumers, especially as many of the cancerogenic or toxic gases elude subjective detection.

Determination of phosphine and other fumigants in air samples by thermal desorption and 2D heart-cutting gas chromatography with synchronous SIM/Scan mass spectrometry and flame photometric detection

Fumigants and volatile industrial chemicals are particularly hazardous to health when a freight container is fumigated or the contaminated material is introduced into its enclosed environment. Phosphine is now increasingly used as a fumigant, after bromomethane--the former fumigant of choice--has been banned by the Montreal Protocol. We have enhanced our previously established thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) method by integrating a second gas chromatographic dimension and a flame photometric detector to allow the simultaneous detection of phosphine and volatile organic compounds (VOCs), providing a novel application. A thermal desorption system is coupled to a two dimensional gas chromatograph using both mass spectrometric and flame photometric detection (TD-2D-GC-MS/FPD). Additionally, the collection of mass spectrometric SIM and Scan data has been synchronised, so only a single analysis is now sufficient for qualitative scanning of the whole sample and for sensitive quantification. Though detection limits for the herewith described method are slightly higher than in the previous method, they are in the low μL m(-3) range, which is not only below the respective occupational exposure and intervention limits but also allows the detection of residual contamination after ventilation. The method was developed for the separation and identification of 44 volatile substances. For 12 of these compounds (bromomethane, iodomethane, dichloromethane, 1,2-dichlorethane, benzene, tetrachloromethane, 1,2-dichloropropane, toluene, trichloronitromethane, ethyl benzene, phosphine, carbon disulfide) the method was validated as we chose the target compounds due to their relevance in freight container handling.

Circulating Mitochondrial DNA as Biomarker Linking Environmental Chemical Exposure to Early Preclinical Lesions Elevation of mtDNA in Human Serum after Exposure to Carcinogenic Halo-Alkane-Based Pesticides

There is a need for a panel of suitable biomarkers for detection of environmental chemical exposure leading to the initiation or progression of degenerative diseases or potentially, to cancer. As the peripheral blood may contain increased levels of circulating cell-free DNA in diseased individuals, we aimed to evaluate this DNA as effect biomarker recognizing vulnerability after exposure to environmental chemicals. We recruited 164 individuals presumably exposed to halo-alkane-based pesticides. Exposure evaluation was based on human biomonitoring analysis; as biomarker of exposure parent halo-methanes, -ethanes and their metabolites, as well as the hemoglobin-adducts methyl valine and hydroxyl ethyl valine in blood were used, complemented by expert evaluation of exposure and clinical intoxication symptoms as well as a questionnaire. Assessment showed exposures to halo alkanes in the concentration range being higher than non-cancer reference doses (RfD) but (mostly) lower than the occupational exposure limits. We quantified circulating DNA in serum from 86 individuals with confirmed exposure to off-gassing halo-alkane pesticides (in storage facilities or in home environment) and 30 non-exposed controls, and found that exposure was significantly associated with elevated serum levels of circulating mitochondrial DNA (in size of 79 bp, mtDNA-79, p = 0.0001). The decreased integrity of mtDNA (mtDNA-230/mtDNA-79) in exposed individuals implicates apoptotic processes (p = 0.015). The relative amounts of mtDNA-79 in serum were positively associated with the lag-time after intoxication to these chemicals (r = 0.99, p<0.0001). Several months of post-exposure the specificity of this biomarker increased from 30% to 97% in patients with intoxication symptoms. Our findings indicate that mitochondrial DNA has a potential to serve as a biomarker recognizing vulnerable risk groups after exposure to toxic/carcinogenic chemicals.

Halogenated hydrocarbon pesticides and other volatile organic contaminants provide analytical challenges in global trading

Protection against infestation of a container cargo by alien species is achieved by mandatory fumigation with pesticides. Most of the effective fumigants are methyl and ethyl halide gases that are highly toxic and are a risk to both human health and the environment. There is a worldwide need for a reliable and robust analytical screening procedure for these volatile chemicals in a multitude of health and environmental scenarios. We have established a highly sensitive broad spectrum mass spectrometry method combined with thermal desorption gas chromatography to detect, identify and quantify volatile pesticide residues. Using this method, 1201 random ambient air samples taken from freight containers arriving at the biggest European ports of Hamburg and Rotterdam were analyzed over a period of two and a half years. This analytical procedure is a valuable strategy to measure air pollution from these hazardous chemicals, to help in the identification of pesticides in the new mixtures/formulations that are being adopted globally and to analyze expired breath samples after suspected intoxication in biomonitoring.

Desensitisation of LH-stimulated cyclic AMP accumulation in isolated bovine luteal cells--effect of phorbol ester.

In this study, we have characterized the LH-mediated desensitisation of receptor-linked cAMP generation in bovine luteal cells. Furthermore, the possibility that protein kinase C could play a role in this process has been investigated. The results obtained, show that the preincubation of Percoll-purified bovine luteal cells with LH diminished the cAMP response during reincubation with LH, depending upon the duration of prior exposure to LH and the concentration of LH used in the first incubation. This desensitisation was specifically dependent upon the prior exposure of the cells to the hormone only, as preincubation with either forskolin or cholera toxin did not result in a desensitised cAMP response to subsequent LH stimulation. On the other hand, LH-desensitised cells retained undiminished responsiveness to restimulation with cholera toxin. Neither the maximum binding capacity nor the affinity of the LH-receptor was affected by exposure of the cells to a desensitising dose of LH. The results demonstrate that in bovine luteal cells, LH produces a homologous desensitisation of the cAMP response which is not mediated by cAMP and that a hormone-receptor interaction appears to be a prerequisite for this process. Preincubation of the cells with varying concentrations of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) did not result in any reduction of LH-induced cAMP response during reincubation. The affinity of LH-receptor was also not affected by PMA pretreatment. In contrast, PMA-pretreated cells consistently produced increased amounts of cAMP when challenged with any of the agonists, LH, cholera toxin or forskolin. The preincubation of the cells with LH in the presence of PMA appears to prevent, at least partially, the desensitising effect of LH. It is concluded that in bovine luteal cells there is no evidence for a role of protein kinase C in LH-induced desensitisation. On the contrary, PMA pretreatment increased the response of adenylate cyclase to a subsequent hormonal stimulation without changing the affinity of the receptors for the hormone. Either an attenuation of the inhibitory N protein or a direct activation of the catalytic unit of adenylate cyclase could be the explanation for the observed effects of PMA. However, available data at present do not offer a choice between the two possibilities.

Lysophosphatidic Acid Antagonizes the Morphoregulatory Effects of the Luteinizing Hormone on Luteal Cells: Possible Role of Small Rho-G-Proteins

Abstract Lysophosphatidic acid (LPA) is a biologically active phospholipid recently introduced as a new marker for ovarian cancer. Because high concentrations of LPA have also been found in the follicular fluid from healthy subjects, one can presume that this biological mediator may have relevance for normal ovarian physiology as well. We have reported earlier that luteal cells possess specific binding sites for LPA. Using these cells as a model, we show now that LPA is able to modulate the morphological cell shape changes induced by LH in that it inhibits the formation of stellate processes induced by LH. This morphoregulatory effect of LPA is mimicked by cytotoxic necrotizing factor 1, a bacterial toxin known to activate small G-proteins from the Rho family. On the other hand, C3-exotransferase that acts mainly through the inhibition of Rho A mimics the effects of LH. Furthermore, we report here that the morphoregulatory effects of LPA are accompanied by the translocation of Rho proteins from the cytosol to cell membrane, an effect generally considered to be an indicator for the activation of Rho-GTPases. During the development and rescue of the corpus luteum, major morphoregulatory effects are exerted by LH that appear to be modulated by LPA via an activation of Rho proteins.

Surprises perilous: Toxic health hazards for employees unloading fumigated shipping containers

The fumigation of freight containers to protect transported goods from fungal and pest infestation has increased worldwide in the last five years due to international regulations requiring fumigation or heat treatment of wooden packaging material and dunnage. We have found in 2008 that every sixth container and its contents do retain harmful concentrations of various fumigants and chemicals, representing a significant health risk for port and transport workers, customs officials, warehousemen, store employees and consumers. The shipping documents of these containers did not provide any information about the fumigation procedure or the used fumigant. We report here the cases of 26 patients introduced to our outpatient clinic with presumed intoxication to fumigants, or with symptoms due to inhaling the air out of fumigated containers. All patients were examined from 2007 to 2010 according to a standardized comprehensive diagnostic program. We were able to confirm the diagnosis based on typical symptoms and extensive clinical examination; by laboratory analysis we identified ethylene dichloride, methyl bromide, phosphine and methylene chloride. The predominant symptoms were headaches, concentration and memory problems, dizziness and nausea, irritation of the skin and mucous membranes and a reduced ability to do exercise. In addition to the neurological and neuropsychological impairments our analyses verified the development of reactive airways dysfunction syndrome (RADS) in 14 of 26 patients with long lasting symptoms due to their contact with fumigants. Intoxications with fumigants are serious and could be avoided. These systematical explored cases show the sustainable impact for health and socio-economic wellbeing. These findings also emphasize the necessity for international standards on permitted fumigants, appropriate labeling in the shipping documents and handling of fumigated containers.