Thomas K. Monsees

Effects of Electromagnetic Fields on Cells: Physiological and Therapeutical Approaches and Molecular Mechanisms of Interaction

This review concentrates on findings described in the recent literature on the response of cells and tissues to electromagnetic fields (EMF). Models of the causal interaction between different forms of EMF and ions or biomolecules of the cell will be presented together with our own results in cell surface recognition. Naturally occurring electric fields are not only important for cell-surface interactions but are also pivotal for the normal development of the organism and its physiological functions. A further goal of this review is to bridge the gap between recent cell biological studies (which, indeed, show new data of EMF actions) and aspects of EMF-based therapy, e.g., in wounds and bone fractures.

Sertoli cells as a target for reproductive hazards

Male fertility can be impaired by various toxicants. Some of them are known to target mainly Sertoli cells, which play an essential role in spermatogenesis. In this study, the in vitro response of immature rat Sertoli cells to various environmental pollutants, including pesticides, oestrogenic compounds and heavy metals, has been investigated. Mitochondrial dehydrogenase activity has been used to measure Sertoli cell viability, while production of lactate and secretion of inhibin B have been used as general and specific cell markers. Sertoli cell viability was not affected after 24‐h exposure to lindane, DDT, ethinyloestradiol or bisphenol A in the concentration range analysed (up to 100, 25 or 50 μm, respectively). In contrast, mercury(II) (EC50=31 μm) and cisplatin (15% decrease in viability at 100 μm) induced some cytotoxic effect. With the exception of the pesticide DDT, all chemicals investigated induced a significant dose‐dependent increase in lactate production after 24‐h exposure to Sertoli cells. Owing to the cytotoxic effect of mercury(II), lactate levels dropped again at concentrations above 20 μm. The pesticide lindane (but not DDT) and both oestrogens significantly increased the production of the Sertoli cell specific hormone inhibin B without affecting cell viability. In contrast, the heavy metals mercury(II) and platinum(II) markedly decreased inhibin B levels. This sharp decrease was already significant at metal concentrations that reduced Sertoli cell viability only moderately (10–15%). In conclusion, the secretion of lactate and inhibin B by immature rat Sertoli cells seems to be a useful and sensitive marker with which to explore potential Sertoli cell toxicants.

Synthesis of fluorochromes and pigments in Malassezia furfur by use of tryptophan as the single nitrogen source

Summary. A new minimal medium consisting only of L‐tryptophan (L‐Trp) and a lipid source induced formation of brown pigmentation only in the species Malassezia furfur, which diffuses into the agar. Strains of the species M. sympodialis and M. pachydermatis failed to grow on this medium. On mDixon medium, however, after replacement of peptone by L‐Trp, growth of all three Malassezia species was achieved. Under these conditions pigment production was observed with all M. furfur strains tested, although the results for M. pachydermatis strains were inconsistent. M. sympodialis strains showed no pigment production. On the minimal medium pigmentogenesis was induced in M. furfur by only 0.01 g% tryptophan; the pH optimum was pH 5. In all M. furfur strains, alternative amino nitrogen sources given concurrently with Trp suppressed pigmentogenesis. Furthermore, there were differences in the optimal temperature among the individual M. furfur strains. CBS 7019, CBS 6000 and CBS 6001 failed to produce pigment at 37°C. The extract of the culture exhibited remarkable fluorescence, and several indole derivatives with a broad spectrum of colours were detected. This finding may have an impact on the clinical appearance of pityriasis versicolor, a very common skin disease caused by lipophilic yeasts of the genus Malassezia. We hypothesize that in pityriasis versicolor metabolic adaptation of Malassezia yeasts to altered nitrogen conditions on superficial skin might be of patho‐physiological importance. Tryptophan as an inducer of pigmentogenesis probably accumulates during excessive sweating, a well‐known manifestation of pityriasis versicolor.

Effects of Different Titanium Alloys and Nanosize Surface Patterning on Adhesion, Differentiation, and Orientation of Osteoblast-Like Cells

To test nanosize surface patterning for application as implant material, a suitable titanium composition has to be found first. Therefore we investigated the effect of surface chemistry on attachment and differentiation of osteoblast-like cells on pure titanium prepared by pulsed laser deposition (TiPLD) and different Ti alloys (Ti6Al4V, TiNb30 and TiNb13Zr13). Early attachment (30 min) and alkaline phosphatase (ALP) activity (day 5) was found to be fastest and highest, respectively, in cells grown on TiPLD and Ti6Al4V. Osteoblasts seeded on TiPLD produced most osteopontin (day 10), whereas expression of this extracellular matrix protein was an order of magnitude lower on the TiNb30 surface. In contrast, expression of the corresponding receptor, CD44, was not influenced by surface chemistry. Thus, TiPLD was used for further experiments to explore the influence of surface nanostructures on osteoblast adhesion, differentiation and orientation. By laser-induced oxidation, we produced patterns of parallel Ti oxide lines with different widths (0.2–10 µm) and distances (2–20 and 1,000 µm), but a common height of only 12 nm. These structures did not influence ALP activity (days 5–9), but had a positive effect on cell alignment. Two days after plating, the majority of the focal contacts were placed on the oxide lines. The portion of larger focal adhesions bridging two lines was inversely related to the line distance (2–20 µm). In contrast, the portion of aligned cells did not depend on the line distance. On average, 43% of the cells orientated parallel towards the lines, whereas 34% orientated vertically. In the control pattern (1,000 µm line distance), cell distribution was completely at random. Because a significant surplus of the cells preferred a parallel alignment, the nanosize difference in height between Ti surface and oxide lines may be sufficient to orientate the cells by contact guiding. However, gradients in electrostatic potential and surface charge density at the Ti/Ti oxide interface may additionally influence focal contact formation and cell guidance.

Local calcium elevation and cell elongation initiate guided motility in electrically stimulated osteoblast-like cells.

Background Investigation of the mechanisms of guided cell migration can contribute to our understanding of many crucial biological processes, such as development and regeneration. Endogenous and exogenous direct current electric fields (dcEF) are known to induce directional cell migration, however the initial cellular responses to electrical stimulation are poorly understood. Ion fluxes, besides regulating intracellular homeostasis, have been implicated in many biological events, including regeneration. Therefore understanding intracellular ion kinetics during EF-directed cell migration can provide useful information for development and regeneration. Methodology/Principal Findings We analyzed the initial events during migration of two osteogenic cell types, rat calvarial and human SaOS-2 cells, exposed to strong (10–15 V/cm) and weak (≤5 V/cm) dcEFs. Cell elongation and perpendicular orientation to the EF vector occurred in a time- and voltage-dependent manner. Calvarial osteoblasts migrated to the cathode as they formed new filopodia or lamellipodia and reorganized their cytoskeleton on the cathodal side. SaOS-2 cells showed similar responses except towards the anode. Strong dcEFs triggered a rapid increase in intracellular calcium levels, whereas a steady state level of intracellular calcium was observed in weaker fields. Interestingly, we found that dcEF-induced intracellular calcium elevation was initiated with a local rise on opposite sides in calvarial and SaOS-2 cells, which may explain their preferred directionality. In calcium-free conditions, dcEFs induced neither intracellular calcium elevation nor directed migration, indicating an important role for calcium ions. Blocking studies using cadmium chloride revealed that voltage-gated calcium channels (VGCCs) are involved in dcEF-induced intracellular calcium elevation. Conclusion/Significance Taken together, these data form a time scale of the morphological and physiological rearrangements underlying EF-guided migration of osteoblast-like cell types and reveal a requirement for calcium in these reactions. We show for the first time here that dcEFs trigger different patterns of intracellular calcium elevation and positional shifting in osteogenic cell types that migrate in opposite directions.

Malassezin--A novel agonist of the arylhydrocarbon receptor from the yeast Malassezia furfur.

The yeast Malassezia furfur converts tryptophan into several indole compounds. One of these, malassezin, was identified as 2-(1H-indol-3-ylmethyl)-1H-indole-3-carbaldehyde (1). It was synthesized from N-Boc-indole-3-carbaldehyde in five steps with 12% overall yield. The compound easily cyclizes to indolo[3,2-b]carbazole (7) which is known to interact with the arylhydrocarbon receptor (AHR). Similarly, malassezin was found to induce cytochrome P450 as an agonist of AHR (EC50 = 1.57 microM) in rat hepatocytes.

Protease-protease inhibitor interactions in Sertoli cell-germ cell crosstalk.

Peritubular cells, Sertoli cells, and germ cells of the seminiferous tubule synthesize and secrete several proteases and protease inhibitors. Experimental evidence suggests that the complex network of proteolytic enzyme activity and their regulation by protease inhibitors play an important role in male reproduction. Interaction between protease and protease inhibitors seems to play an important role in remodeling and restructuring of the seminiferous tubule during spermatogenesis. Controlled proteolytic activity is also involved in the migration of germ cells from the basal compartment to the lumen of the seminiferous epithelium, and in the release of spermatids during spermiation. The recently reported occurrence of Sertoli cell membrane-associated proteases indicate the possible involvement of regulatory peptide systems within the testis. This view is supported by the detection of all components of one of these paracrine systems, the kallikrein-kinin system, in cells of the seminiferous tubule.

Possible involvement of proteases in the regulation of spermatogenesis

Summary Proteolytic enzymes, which are synthesized and secreted by cells of the seminiferous tubule of the testis, have important functions in spermatogenesis. We performed metabolic studies using small peptide hormones as a substrate to investigate the activity of proteases in cultured Sertoli cells of the rat. High‐performance liquid chromatographic analysis of the cell culture supernatants showed cleavage of met‐ and leu‐enkephalin, substance P, and bradykinin. No peptidolysis was observed for the cyclic peptide oxytocin. The hormone cleavage pattern and the use of specific protease inhibitors in peptide degradation experiments demonstrated activities of several proteases in Sertoli cells. These are mainly metalloproteinases including neutral metalloendo‐peptidases, angiotensin‐converting enzyme and aminopeptidases. In addition, activities of serine and aspartic proteases were detected. Only marginal proteolytic activities were observed in Sertoli cell conditioned supernatants, indicating that the investigated proteases are mainly located on Sertoli cell membranes. The peptide hormones used in this study have been found to play a potential role in the endocrine, paracrine or autocrine regulation of testicular cells. The membrane‐associated proteases reported here may therefore be involved in the metabolism and inactivation of these peptides.

Elements of the kallikrein–kinin system are present in rat seminiferous epithelium

Peptide hormones are involved in the paracrine regulation of several physiological processes. A possible function of the kallikrein-kinin system (KKS) in mammalian reproduction has been discussed. To evaluate its putative role in spermatogenesis, we searched for components of the KKS (kallikrein, kininases, kinin receptor) in the rat testis. Specific immunostaining demonstrated that the kininogenase tissue kallikrein was present in round and elongated spermatids. Leydig cells, Sertoli cells, peritubular cells, spermatogonia and spermatocytes were not stained. Bradykinin in the supernatant of Sertoli cell cultures was effectively degraded. The resulting metabolites were analysed by high-performance liquid chromatography (HPLC). Specific protease inhibition in the degrading experiments confirmed the occurrence of several metalloproteases on Sertoli cell membranes, including neutral metalloendopeptidases (NEP 24.11 and NEP 24.15), kininase type II (angiotensin converting enzyme, ACE), and kininase type I (metallocarboxypeptidase). Northern blots hybridized with a bradykinin B2 receptor probe showed the presence of B2 receptor mRNA in testis homogenate and Sertoli cell extract. All components of the kallikrein-kinin system are present within the seminiferous epithelium of the rat. Therefore, this paracrine peptide system may play a role in the regulation of Sertoli cell function or in the Sertoli cell-germ cell crosstalk.

Influence of gossypol on the secretory function of cultured rat sertoli cells.

Cottonseed gossypol is a potent male contraceptive in several mammalian species including man. Sertoli cells play a crucial role in spermatogenesis. Therefore, the antifertility competence of gossypol may reflect a change in Sertoli cell function. Rat primary cultures were used to examine the effect of gossypol on cell viability, mitochondrial dehydrogenase function, lactate production and secretion of the Sertoli cell-specific protein inhibin. Exposure for 24 h to gossypol (3-6 microM) significantly enhance secretion of lactate but reduce secretion of inhibin without affecting cell viability. At 9-15 microM, the observed decrease of both lactate and inhibin accumulation apparently resulted from Sertoli cell degeneration and death, because viability and mitochondrial function were also reduced. The results suggest that mitochondria of Sertoli cells are a possible target for gossypol-induced infertility.