The effect of hydrocortisone and methylphenidate on the growth of murine L929 fibroblast and chick brain neuronal cells

Abstract

Most of the medicines used to treat children have not been fully developed and assessed. Therefore it is not paradoxically that most of the catastrophes observed with medicines in the 20th century happened in children. Children are not small adults; their pharmacokinetics and pharmacodynamics differ for each developmental stage. Growth and maturation processes, as well as certain specific diseases are unique to children. Specific consequences of medical interventions may be seen in children and may only appear long after exposure. Increasing concern is raised regarding drug-induced congenital malformations and drug-drug interactions in children and pregnant women. Little toxicological information regarding prenatal exposure is available. Medicinal products for children need to be tested scientifically to be of significant clinical value. Aim of this study was to determine the combined and separate effects of corticosteroids (in particular hydrocortisone (HC)) and psychostimulants (in particular methylphenidate hydrochloride (MPH)) on cellular function, brain development and skeletal growth. Cytotoxicity was determined in an established L929 cell line and successful cultured primary neuron and limbbud, vertebrae micromass cell cultures. The cultures were prepared and increasing concentrations of HC (0, 3.98, 23.88, 143.3, 859.9 and 5159ìM) or/and MPH (0, 0.57, 3.42, 20.55, 123, 740ìM) were added. The effect of HC and MPH separately and in combination was tested using different bioassays such as the MTT, NR, CV and MB bioassay, which measures changes in cell viability, lysosome membrane integrity, cell number, and RNA and DNA content respectively. The results of all four bioassays showed HC to be toxic; with increasing concentration of HC (in 23.88ìM to 5159ìM). However, the mechanism of toxicity is still unclear. HC demonstrate mitogenic properties in low concentrations in the neuron culture. In contrast MPH also administered in the ìM showed only toxicity at 740ìM. When HC and MPH were administered together to cultures, HC was found to exhibit its cytotoxicity in the presence of MPH. MPH showed no synergistic, additive or cyto-protective ability. A difference in cell morphology was observed after cytotoxic insult with HC or HC combined with MPH. There are essentially four potential targets of drug action: nucleic acids, specific enzymes, microtubules, and hormone/growth factor receptors and it seems that HC and MPH have different mechanisms of action in targeting a cell. The chick embryo has served as a model for investigating normal cell differentiation, human neural tube defects, foetal alcohol syndrome, and other developmental and teratogenic conditions. Therefore the chorioallantoic membrane (CAM) provided a suitable in vivo developmental model, in which prenatal exposure to HC and MPH separately and combined was studied. In this study HC and MPH were injected onto the CAM on EE4 and EE6 of development and evaluated at EE8 or EE13. To investigate developmental toxicity and associated mechanisms, chick embryo weight, viability, morphology, abnormalities and skeletal growth were assessed. Whole legs were stained for bone and cartilage with Alcian Blue/Alizarin Red. MPH was found to increase mortality at high dosages (18.5nmol) after first exposure at EE4. The chick embryos where exposed to 0.138ìmol and 0.685ìmol HC in the first set of experiments followed by 0.066ìmol and 0.329ìmol HC in the second set of experiments. HC was found to significantly decrease body weight and viability, especially after the second exposure at EE6. Increased abnormalities were observed in the chick embryo group exposed to low concentration (0.138ìmol) HC, where the viability of chick embryos were higher. Abnormalities included abnormal anterior thoracic walls with ectopic hearts. Most abnormalities were observed in the dead embryos. Due to early embryonic death of embryos, skeletal abnormalities were rare. It is therefore believe that MPH is not toxic except at high concentrations, but that HC is toxic even at low concentrations. Although cell death and morphological changes could be detected, the exact dosages which can be used to extrapolate toxicity in children, need to be further investigated. This study provides a step towards progressing the well-being and treatment, prevention and diagnosis of ill health in children. Copyright