Important issues in developmental toxicity testing
Studies of individual development and its possible deterioration have been the concern since the 19th century, when Etienne Geoffroy de Saint-Hilaire (1772-1844) with his pioneer experiments opened the door for future experimental teratologists. Later scientists, focused on environmental agents which can alter embryonic and fetal development, such hyperthermia, malnutrition, pharmaceuticals, microbial toxins etc. Although the history of teratology involves many notable scientists, it has gained prominence after the big thalidomide tragedy in 1961. Principles of teratology were proposed later by James Wilson in his monograph Environment and Birth Defects (Wilson, 1973).
Developmental origin of chronic diseases: toxicological implication
Human epidemiological and experimental animal studies show that suboptimal environments in fetal and neonatal life exerts a profound influence on physiological function and risk of disease in adult life. The molecular, cellular, metabolic, endocrine and physiological adaptations to intrauterine nutritional conditions result in permanent alterations of cellular proliferation and differentiation of tissues and organ systems, which in turn can manifest by pathological consequences or increased vulnerability to chronic diseases in adulthood. Intrauterine growth restriction (IUGR) due to intrauterine development derangements is considered the important factor in development of such diseases as essential hypertension, diabetes mellitus, ischemic diseases of the heart, osteoporosis, respiratory, neuropsychiatric and immune system diseases.
An early life exposures to dietary and environmental exposures can have a important effect on epigenetic code, resulting in diseases developed later in life. The concept of the "developmental programming" and Developmental Origins of Adult Diseases (DOHaD) has become well accepted because of the compelling animal studies that have precisely defined the outcomes of specific exposures. The environmental pollullutants and other chemical toxicants may influence crucial cellular functions during critical periods of fetal development and permanently alter the structure or function of specific organ systems. Developmental epigenetics is believed to establish "adaptive" phenotypes to meet the demands of the later-life environment. Resulting phenotypes that match predicted later-life demands will promote health, while a high degree of mismatch will impede adaptability to later-life challenges and elevate disease risk. The rapid introduction of synthetic chemicals, environmental pollutants and medical interventions, may result in conflict with the programmed adaptive changes made during early development, and explain the alarming increases in some diseases.
Metabolic syndrome belongs to the most important risk factors of cardiovascular diseases. The aim of this study was to investigate changes in cardiovascular system induced by high cholesterol and high fat diet (HCHF) in HTG rats and their influence by a pyridoindole antioxidant – SMe1EC2 (S). The effects of S were compared with those of atorvastatin (A). Male HTG rats were fed HCHF (1% cholesterol + 7.5% lard) for 4 weeks. S and A were administered p.o., 50 mg/kg b.w. Following experimental groups were used: Wistar rats (W), hypertriglyceridemic rats (HTG), HTG rats fed HCHF (CHOL), HTG+S (S-HTG), CHOL+S (S-CHOL), and CHOL+A (A-CHOL). Values of blood pressure (BP) and selected ECG parameters were monitored in conscious animals, functions of the isolated heart and aorta were analyzed ex vivo. At the end of the experiment, systolic (sBP) and diastolic (dBP) blood pressure was increased in HTG and CHOL. S and A decreased BP in all treated groups. Accordingly with BP changes, the aortic endothelial function of CHOL was damaged. Both S and A administration ameliorated the endothelium-dependent relaxation to values of W. PQ and QTc intervals were prolonged in CHOL, while the treatment with S or A improved ECG findings. Prodysrhythmogenic threshold was decreased significantly in CHOL and both treatments returned it to the control values. In conclusion, HCHF increased BP, impaired endothelial relaxation of the aorta and potentiated susceptibility of myocardium to dysrhythmias. The effect of S on the changes induced by HCHF diet was more pronounced than that of A.
Elevated plasma cholesterol, especially low density lipoprotein (LDL) cholesterol, is one of the major risk factors for atherosclerosis and coronary heart disease. Hereditary hypertriglyceridemic rats (hHTG) were developed as a new inbred model for the study of relationships between blood pressure and metabolic abnormalities. The aim of this work was to determine the cholesterol-lowering and antioxidant effects of the novel pyridoindol derivative SMe1EC2, compared to the cholesterol-lowering drug atorvastatin, in rats fed either standard or high-fat and high-cholesterol diet (HFC; 1% cholesterol and 7.5% lard fat). Male hHTG rats fed HFC (HTG+HFC) were administered with SMe1EC2 or atorvastatin (both 50 mg/kg/day p.o.) for 4 weeks. Physiological status of animals was monitored by the measurement of preprandial glucose levels and blood pressure. Lipid profile was characterized by the serum levels of total cholesterol (TC), HDL-, LDL-cholesterol and triglycerides (TRG). The concentration of thiobarbituric acid reactive substances (TBARS) was evaluated in the kidney, liver and serum. Further, the assessment of pro-inflammatory cytokines TNF-α, IL-1 and IL-6 in the serum was completed. Feeding the animals with HFC diet resulted in increased serum levels of TC, LDL and TRG. SMe1EC2 ameliorated serum levels of LDL in hHTG rats, both on standard and HFC diet. These effects were comparable with those of the standard hypolipidemicum atorvastatin. SMe1EC2 lowered blood pressure, tissue TBARS concentrations and serum IL-1 levels of HTG+HFC rats. Beneficial effects together with very good toxicity profile predestinate SMe1EC2 to be promising agent for further surveys related to metabolic syndrome features.
Synthetic pyridoindole-type substances derived from the lead compound stobadine represent promising agents in treatment of a range of pathologies including neurological disorders. The beneficial biological effects were suggested to be likely associated with their capacity to ameliorate oxidative damage.
In our study, the effect of supplementation with the derivative of stobadine, SMe1EC2, on ageing-related cognitive decline in rats was investigated. The 20-months-old male Wistar rats were administered SMe1EC2 at a low dose, 0.5 mg/kg, daily during eight weeks. Morris water maze test was performed to assess the spatial memory performances. The cell-based assays of capacity of SMe1EC2 to modulate proinflammatory generation of oxidants by microglia were also performed.
The rats treated with SMe1EC2 showed significantly increased path efficiency, significantly shorter time interval of successful trials and exerted also notably lower frequencies of clockwise rotations in the pool compared to non-supplemented aged animals. Mildly improved parameters included test durations, distances to reach the platform, time in periphery of the pool and overall rotations in the water maze. However, the pyridoindole SMe1EC2 did not show profound inhibitory effect on production of nitric oxide and superoxide by activated microglial cells.
In conclusion, our study suggests that pyridoindole SMe1EC2, at low doses administered chronically, can act as cognition enhancing agent in aged rats. The protective mechanism less likely involves direct modulation of proinflammatory and prooxidant state of microglia, the prominent mediators of neurotoxicity in brain ageing and neurodegeneration.
Protection of the vascular endothelium in experimental situations
One of the factors proposed as mediators of vascular dysfunction observed in diabetes is the increased generation of reactive oxygen species (ROS). This provides support for the use of antioxidants as early and appropriate pharmacological intervention in the development of late diabetic complications. In streptozotocin (STZ)-induced diabetes in rats we observed endothelial dysfuction manifested by reduced endothelium-dependent response to acetylcholine of the superior mesenteric artery (SMA) and aorta, as well as by increased endothelaemia. Changes in endothelium-dependent relaxation of SMA were induced by injury of the nitric oxide radical (·NO)-signalling pathway since the endothelium-derived hyperpolarising factor (EDHF)-component of relaxation was not impaired by diabetes. The endothelial dysfunction was accompanied by decreased ·NO bioavailabity as a consequence of reduced activity of eNOS rather than its reduced expression. The results obtained using the chemiluminiscence method (CL) argue for increased oxidative stress and increased ROS production. The enzyme NAD(P)H-oxidase problably participates in ROS production in the later phases of diabetes. Oxidative stress was also connected with decreased levels of reduced glutathione (GSH) in the early phase of diabetes. After 10 weeks of diabetes, adaptational mechanisms probably took place because GSH levels were not changed compared to controls. Antioxidant properties of SMe1EC2 found in vitro were partly confirmed in vivo. Administration of SMe1EC2 protected endothelial function. It significantly decreased endothelaemia of diabetic rats and improved endothelium-dependent relaxation of arteries, slightly decreased ROS-production and increased bioavailability of ·NO in the aorta. Further studies with higher doses of SMe1EC2 may clarify the mechanism of its endothelium-protective effect in vivo.