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Mutagen-induced variations of the cellular DNA content have been studied in mouse bone marrow cells in uiuo using high resolution flow cytometric techniques. During the first days after a single injection of the chemical mutagen cyclophosphamide an increased coefficient of variation in the GI peak of the flow histograms was observed. The magnitude and duration of this effect were dose-dependent. The reproducibility of the measurements was high, indicating that individual variability between animals and instrumental dispersion is small. The results demonstrate that on the basis of the flow cytometric measurement of cellular DNA content, a short-term in uiuo test for mutagenicity can be established which is much faster than conventional cytogenetic methods.Key terms: Mouse bone marrow, chemical mutagens, flow cytometry, DNA content variations, DNA staining method, mutagenicity testing.The need to develop rapid, reproducible and reliable methods for the detection of mutagens has been increasingly felt in recent years because of enhancement in environmental polstructural chromosome aberrations and dysfunction of the mitotic apparatus can cause an unequal distribution of the DNA in the daughter cells. It has been shown that by using high resolution flow cytometric techniques an increased dispersion Of the DNA 'Ontent produced in this way can be measured in X-irradiated Chinese hamster cells zn uztro access to food and drinking water. Cyclophosphamide (analytical grade, (Heidelberg, Germany) No. 17681) was administered by a single ip injection (0.56-0.7 ml of 0.075-0.6% solution in physiological saline). Control animals received an equivalent volume of saline. After cells, harvested by flushing out the femur with 5 ml of Ca-and Mgfree phosphate buffered saline containing 0.5 mM EDTA disodium salt, were fixed in 70% ethanol and stored under refrigeration.For accurate and reproducible measurements of DNA content a procedure based on a method described by Pinaev et al, 15) was developed. Fixed cells were pelleted by centrifugation at 200 X g for 10 min, resuspended at a concentration of 2-to 6 x 10" cells in 1 ml lutants. In a proliferating population, mutagen-induced different intervals of time, mice were sacrificed and bone m m o w and mouse bone marrow cells in uiuo (4). Hopefully, the flow cytometric measurement of the dispersion in the cellular DNA content induced by interactions with environmental agents can provide a powerful tool for cytogenetic investigations and mutagenicity testing. In the present work, time dependence, dose-effect relationship and reproducibility of DNA dispersion induced by the chemical mutagen cyclophosphamide were studied in mice. Materials and MethodsMale NMRI mice weighing 28-35 g were used in these experiments. The animals were maintained in standard plastic boxes and had free of 0.2 M citric acid at pH 1.8 with 0.5% Tween 20, and incubated at room temperature for 20 min. Subsequently, the pH was raised to 7.4 and the cells were stained by addition of 9 ml of disodium hydrogen phosphate...

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Previous in vitro studies have demonstrated that endogenously produced human interleukin‐8 (IL‐8) can act as an important growth factor for human melanoma cells in vitro. The present study, has investigated whether IL‐8 mRNA expression in primary melanomas may be of prognostic relevance with regard to melanoma progression and metastatic spread. In order to evaluate the clinical significance of IL‐8 mRNA expression of melanoma cells in vivo, 59 melanocytic tissue specimens (37 primary melanomas and 22 melanocytic naevi) were studied using a semiquantitative in situ hybridization technique. Significant mRNA expression of IL‐8 was found in 59 per cent (22/37) of melanomas. In 19 per cent (7/37) of the malignant melanomas, additional hybridization signals were noted within keratinocytes of the overlying epidermis. In contrast, paralesional normal‐appearing epidermis and melanocytes in non‐malignant lesions (melanocytic naevi) showed no IL‐8 mRNA. Analysis of the relationship between IL‐8 expression and clinico‐histopathological features showed a significant association between IL‐8 mRNA expression and the histological melanoma subtype (IL‐8 mRNA: 14/19 in superficial spreading melanoma versus 4/12 in nodular melanoma, p< 0·05). Furthermore, IL‐8 expression in primary tumours could be correlated with the patients' clinical course, with time to progression being significantly reduced in primary tumours expressing IL‐8 in either the tumour cells or keratinocytes of the overlying epidermis. These results demonstrate for the first time that IL‐8 expression, as detected by in situ hybridization in primary tumours, may serve as a significant prognostic factor for tumour progression in human malignant melanoma. Copyright © 1999 John Wiley & Sons, Ltd.