Toxicity as prime selection criterion among SARS-active herbal medications.

We present here a new selection criterion for prioritizing research on efficacious drugs for the fight against COVID-19: the relative toxicity versus safety of herbal medications, which were effective against SARS in the 2002/2003 epidemic. We rank these medicines according to their toxicity versus safety as basis for preferential rapid research on their potential in the treatment of COVID-19. The data demonstrate that from toxicological information nothing speaks against immediate investigation on, followed by rapid implementation of Lonicera japonica, Morus alba, Forsythia suspensa, and Codonopsis spec. for treatment of COVID-19 patients. Glycyrrhiza spec. and Panax ginseng are ranked in second priority and ephedrine-free Herba Ephedrae extract in third priority (followed by several drugs in lower preferences). Rapid research on their efficacy in the therapy - as well as safety under the specific circumstances of COVID-19 - followed by equally rapid implementation will provide substantial advantages to Public Health including immediate availability, enlargement of medicinal possibilities, in cases where other means are not successful (non-responders), not tolerated (sensitive individuals) or just not available (as is presently the case) and thus minimize sufferings and save lives. Moreover, their moderate costs and convenient oral application are especially advantageous for underprivileged populations in developing countries.

Differential p38-dependent signalling in response to cellular stress and mitogenic stimulation in fibroblasts.

p38 MAP kinase is known to be activated by cellular stress finally leading to cell cycle arrest or apoptosis. Furthermore, a tumour suppressor role of p38 MAPK has been proposed. In contrast, a requirement of p38 for proliferation has also been described. To clarify this paradox, we investigated stress- and mitogen-induced p38 signalling in the same cell type using fibroblasts. We demonstrate that - in the same cell line - p38 is activated by mitogens or cellular stress, but p38-dependent signalling is different. Exposure to cellular stress, such as anisomycin, leads to a strong and persistent p38 activation independent of GTPases. As a result, MK2 and downstream the transcription factor CREB are phosphorylated. In contrast, mitogenic stimulation results in a weaker and transient p38 activation, which upstream involves small GTPases and is required for cyclin D1 induction. Consequently, the retinoblastoma protein is phosphorylated and allows G1/S transition. Our data suggest a dual role of p38 and indicate that the level and/or duration of p38 activation determines the cellular response, i.e either proliferation or cell cycle arrest.

Circadian rhythms and chemical carcinogenesis: Potential link. An overview.

Circadian rhythm is an integral and not replaceable part of the organism's homeostasis. Its signalling is multidimensional, overlooking global networks such as chromatin remodelling, cell cycle, DNA damage and repair as well as nuclear receptors function. Understanding its global networking will allow us to follow up not only organism dysfunction and pathology (including chemical carcinogenesis) but well-being in general having in mind that time is not always on our side.

In vivo fate mapping with SCL regulatory elements identifies progenitors for primitive and definitive hematopoiesis in mice.

One of the principal issues facing biomedical research is to elucidate developmental pathways and to establish the fate of stem and progenitor cells in vivo. Hematopoiesis, the process of blood cell formation, provides a powerful experimental system for investigating this process. Here, we employ transcriptional regulatory elements from the stem cell leukemia (SCL) gene to selectively label primitive and definitive hematopoiesis. We report that SCL-labelled cells arising in the mid to late streak embryo give rise to primitive red blood cells but fail to contribute to the vascular system of the developing embryo. Restricting SCL-marking to different stages of foetal development, we identify a second population of multilineage progenitors, proficient in contributing to adult erythroid, myeloid and lymphoid cells. The distinct lineage-restricted potential of SCL-labelled early progenitors demonstrates that primitive erythroid cell fate specification is initiated during mid gastrulation. Our data also suggest that the transition from a hemangioblastic precursors with endothelial and blood forming potential to a committed hematopoietic progenitor must have occurred prior to SCL-marking of definitive multilineage blood precursors.

Role of cAMP in mediating AHR signaling.

Regulation of the nuclear import of many transcription factors represents a step in gene regulation which is crucial for a number of cellular processes. The aryl hydrocarbon receptor (AHR), a basic helix-loop-helix protein of the PAS (PER-ARNT-SIM) family of transcriptional regulators is a cytosol-associated and ligand-activated receptor. The environmental toxin dioxin binds with high affinity to AHR rendering it nuclear and leading to the activation of AHR sensitive genes. However, the fact, that the AHR mediates a large variety of physiological events without the involvement of any known exogenous ligand, including liver and vascular system development, maturation of the immune system, regulation of genes involved in cellular growth, cell differentiation and circadian rhythm, speaks for an important role of AHR in cell biology independent of the presence of an exogenous ligand. Different approaches were applied to study mechanism(s) which render AHR nuclear and design its function in absence of exogenous ligands. We found that AHR is sensitive to cAMP signaling mediated by cAMP-dependent protein kinase (PKA) which fundamentally differs from AHR signaling mediated by the exogenous ligand dioxin. It has been shown that PKA mediated signaling can be confined by compartmentalization of signaling components in microdomains conferring specificity to signaling by the ubiquitous second messenger cAMP. Moreover, A-kinase-anchoring proteins (AKAPs) and newly discovered cAMP receptors, Epac (exchange protein directly activated by cAMP), may give us a further chance to enter into new dimensions of cAMP signal transmissions that potentially may bring us closer to AHR physiology.

Environmental exposure to dioxins and polychlorinated biphenyls reduce levels of gonadal hormones in newborns: results from the Duisburg cohort study.

BACKGROUND: Endocrine dysfunction related to the hypothalamic-pituitary-thyroid (HPT) and/or the hypothalamic-pituitary-gonadal axis (HPG) is being discussed as underlying developmental adversity of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs). This study was done to evaluate effects related to the HPG axis. METHODS: A birth-cohort study was initiated in the year 2000. Healthy mother-infant pairs were recruited in the industrialized city of Duisburg, Germany. Dioxins, dioxin-like PCBs and six indicator PCBs were measured in maternal blood during pregnancy and in maternal milk. Testosterone and estradiol levels were measured in maternal and cord serum of 104 mother-infant pairs representing a subsample with a complete data set of the total basic sample of 232 participants. Linear regression analysis was used to describe the association of PCDD/Fs or PCB in maternal blood or milk with sex steroid concentrations after adjustment for confounding. RESULTS: Median concentrations for PCDD/Fs in maternal blood fat and milk fat in terms of WHO-TEq were 15.3 and 13.1pg WHO-TEq/g, respectively, and for the sum of the indicator PCBs (#28, #52, #101, #138, #153, #180) 149 and 177ng/g. The adjusted ratio of geometric means when doubling the concentration of PCDD/Fs in maternal blood fat was 0.86, 95% confidence interval (95% CI): 0.72-1.03 for testosterone and 0.73 (0.61-0.87) for estradiol in cord serum. Typically, testosterone reduction was more pronounced in cord serum of female and estradiol reduction in that of male babies. Reduction of hormone levels was generally more pronounced for dioxins than for indicator PCBs. CONCLUSIONS: The hypothalamic-pituitary-gonadal axis of newborn babies is influenced by prenatal exposure to PCDD/Fs and PCBs in a manner suggestive of AhR-mediation. The clinical relevance of this finding remains to be established, however.

Drug-metabolizing enzymes in the skin of man, rat, and pig.

The mammalian skin has long been considered to be poor in drug metabolism. However, many reports clearly show that most drug metabolizing enzymes also occur in the mammalian skin albeit at relatively low specific activities. This review summarizes the current state of knowledge on drug metabolizing enzymes in the skin of human, rat, and pig, the latter, because it is often taken as a model for human skin on grounds of anatomical similarities. However only little is known about drug metabolizing enzymes in pig skin. Interestingly, some cytochromes P450 (CYP) have been observed in the rat skin which are not expressed in the rat liver, such as CYP 2B12 and CYP2D4. As far as investigated most drug metabolizing enzymes occur in the suprabasal (i.e. differentiating) layers of the epidermis, but the rat CYP1A1 rather in the basal layer and human UDP-glucuronosyltransferase rather in the stratum corneum. The pattern of drug metabolizing enzymes and their localization will impact not only the beneficial as well as detrimental properties of drugs for the skin but also dictate whether a drug reaches the blood flow unchanged or as activated or inactivated metabolite(s).

Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system.

BACKGROUND: Conditional gene regulatory systems ensuring tight and adjustable expression of therapeutic genes are central for developing future gene therapy strategies. Among various regulatory systems, tetracycline-controlled gene expression has emerged as a safe and reliable option. Moreover, the tightness of tetracycline-regulated gene switches can be substantially improved by complementing transcriptional activators with antagonizing repressors. METHODS: To develop novel tetracycline-responsive transcriptional repressors, we fused various transcriptional silencing domains to the TetR (B/E) DNA-binding and dimerization domain of the Tn10-encoded tetracycline resistance operon (TetR (B/E)). The resulting fusion proteins were individually tested for their ability to repress transcription of the constitutively active hypoxanthine phosphoribosyltransferase (HPRT) promoter. In addition, compatibility with the commonly used reverse tetracycline-controlled transactivator system (rtTA-system) and responsiveness to the pharmacological effector doxycycline (DOX) were evaluated. Finally, inducibility, effector-dependent promoter activity and the modification of histone H3 and H4 of the active versus the repressed target promoter were determined. RESULTS: Fusion of the human deacetylase 4 (HDAC4) carboxy-terminal silencing domain to TetR (B/E) resulted in a functional transcriptional repressor. This novel repressor, termed tTS-H4, efficiently reduced the activity of the murine HPRT promoter and a constitutively active human cytomegalovirus (hCMV) minimal promoter. Furthermore, combining tTS-H4 with the rtTA transcriptional activator allowed for grading, turning off and resuming target gene expression over several orders of magnitude without background. CONCLUSIONS: The tTS-H4 repressor is compatible with the commonly used rtTA transcriptional activation system and is a versatile new tool for tightly and adjustably regulating conditional gene expression.

Styrene metabolism, genotoxicity, and potential carcinogenicity.

This report reviews styrene biotransformation, including minor metabolic routes, and relates metabolism to the genotoxic effects and possible styrene-related carcinogenicity. Styrene is shown to require metabolic activation in order to become notably genotoxic and styrene 7,8-oxide is shown to contribute quantitatively by far the most (in humans more than 95%) to the genotoxicity of styrene, while minor ring oxidation products are also shown to contribute to local toxicities, especially in the respiratory system. Individual susceptibility depending on metabolism polymorphisms and individual DNA repair capacity as well as the dependence of the nonlinearity of the dose-response relationships in the species in question and the consequences for risk evaluation are analyzd.

Aryl hydrocarbon receptor activation by cAMP vs. dioxin: divergent signaling pathways.

Even before the first vertebrates appeared on our planet, the aryl hydrocarbon receptor (AHR) gene was present to carry out one or more critical life functions. The vertebrate AHR then evolved to take on functions of detecting and responding to certain classes of environmental toxicants. These environmental pollutants include polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene), polyhalogenated hydrocarbons, dibenzofurans, and the most potent small-molecular-weight toxicant known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). After binding of these ligands, the activated AHR translocates rapidly from the cytosol to the nucleus, where it forms a heterodimer with aryl hydrocarbon nuclear translocator, causing cellular responses that lead to toxicity, carcinogenesis, and teratogenesis. The nuclear form of the activated AHR/aryl hydrocarbon nuclear translocator complex is responsible for alterations in immune, endocrine, reproductive, developmental, cardiovascular, and central nervous system functions whose mechanisms remain poorly understood. Here, we show that the second messenger, cAMP (an endogenous mediator of hormones, neurotransmitters, and prostaglandins), activates the AHR, moving the receptor to the nucleus in some ways that are similar to and in other ways fundamentally different from AHR activation by dioxin. We suggest that this cAMP-mediated activation may reflect the true endogenous function of AHR; disruption of the cAMP-mediated activation by dioxin, binding chronically to the AHR for days, weeks, or months, might be pivotal in the mechanism of dioxin toxicity. Understanding this endogenous activation of the AHR by cAMP may help in developing methods to counteract the toxicity caused by numerous environmental and food-borne toxic chemicals that act via the AHR.

Guanine 6-O-methylation pattern within the dioxin responsive element of the CYP1A1 enhancer shows two critical guanines for AhR/ARNT binding.

The core-recognition motif for TCDD-liganded AhR/ARNT complex of the dioxin-responsive element (DRE) contains four guanine residues, three on the antisense (5'-T(T)/(A)GCGTG-3') and one on the sense (5'-CACGC(A)/(T)A-3') strand. It has been reported that, in methylation-protection and methylation-interference assays, the TCDD-liganded AhR/ARNT contacts all four guanine residues. On the other hand, it is known that some anticancer drugs, and various environmental and workplace chemicals, including strongly human carcinogenic nitrosamines, lead to the highly miscoding 6-O-methylation of guanine. In the present study, we have investigated whether specific methylation of guanine at the 6-O-position interferes with the binding of TCDD-liganded AhR/ARNT complex to its recognition motif in the CYP1A1 enhancer, and how individual 6-O-methylated guanines contribute to this interference. We found that only two 6-O-methylguanine residues are critical: the closest to the 5'-end within the three-nucleotide sequence (5'-GTG-3'), identical to a half-site E-box element, on the antisense strand, and the only guanine on the sense strand. In contrast, the 6-O-methylguanine closest to the 5'-end (well) and the one closest to the 3'-end (to a lesser extent), both on the antisense strand, were still able to bind the TCDD-liganded AhR/ARNT complex. This shows that the 6-O-methylation of guanine in the core sequences of CYP1A1 enhancer interferes with the binding of the ligand-activated AhR/ARNT complex in a differentially selective manner; it fully impedes binding of this complex to DRE (the prerequisite of most of the toxic effects of TCDD) only when one of the two particular guanines is methylated at the 6-O-position.

Cytochrome-P450 phosphorylation as a functional switch.

Xenobiotic metabolizing cytochromes P450 (CYP) were shown to be phosphorylated in vitro (using purified protein kinases together with purified CYPs), in intact cells (in V79 cells after transfection of cDNAs coding for individual CYPs, in diagnostic mutants, in hepatocytes), and in whole organisms (rats). CYP phosphorylation is highly isoenzyme selective in that only some CYPs are phosphorylated. Protein kinase A (PKA) was identified as a major catalyst for the phosphorylation of CYPs. The PKA recognition motif Arg-Arg-X-Ser is present in several members of the CYP2 family, but is used by only some of them, most notably by CYP2B1/2B2 and CYP2E1. For CYP2B1 it was shown that a substantial portion but not the entire pool of CYP2B1 molecules is phosphorylated and that the phosphorylated portion is catalytically fully inactive. Phosphorylation of CYPs is a very fast process (visible at the earliest time point experimentally investigated after introduction of phosphorylation-supporting measures, which was 2.5min) and the phosphorylated protein is immediately inactive (i.e., the time curves of phosphorylation and inactivation are superimposable). Thus in contrast to the slower process controlling CYP activities by enzyme induction, CYP phosphorylation controls CYP function like a switch. The physical entity of the switch was identified by site-directed mutation as the phosphoryl acceptor Ser in the PKA recognition motif, which is Ser(138) in CYPs 2B (rat CYP2B1 and rabbit CYP2B4) and its homologous Ser(139) in CYP2E1. The function of this switch was demonstrated for the drastic changes in the control of the genotoxic metabolites of mutagenic carcinogens as well as for the control of effectiveness versus unwanted toxicity of cytostatic cancer drugs.

Fast regulation of cytochrome P450 activities by phosphorylation and consequences for drug metabolism and toxicity.

In contrast to the well-known regulation of cytochrome P450 (CYP) activity by enzyme induction, which represents a process with slow onset and slow offset, more recent studies revealed phosphorylation as a fast (within observation instantaneous) and isoenzyme-selective regulation. The phosphorylated enzyme (investigated isozyme: CYP2B1) was fully inactive. The phosphorylation is mediated by PKA and hence under control of hormones and drugs that alter cellular cAMP levels. The consequences for the metabolic control of toxic species derived from drugs and environmental carcinogens are discussed. This information will help to improve therapy with drugs metabolized by CYPs which are phosphorylated by PKA, especially if these drugs possess a narrow window between required effectiveness and unacceptable toxicity.