Programming of metabolic effects in C57BL/6JxFVB mice by in utero and lactational exposure to perfluorooctanoic acid.

Perfluorooctanoic acid (PFOA) is known to cause developmental toxicity and is a suggested endocrine disrupting compound (EDC). Early life exposure to EDCs has been implicated in programming of the developing organism for chronic diseases later in life. Here we study perinatal metabolic programming by PFOA using an experimental design relevant for human exposure. C57BL/6JxFVB hybrid mice were exposed during gestation and lactation via maternal feed to seven low doses of PFOA at and below the NOAEL used for current risk assessment (3-3000 µg/kg body weight/day). After weaning, offspring were followed for 23-25 weeks without further exposure. Offspring showed a dose-dependent decrease in body weight from postnatal day 4 to adulthood. Growth under high fat diet in the last 4-6 weeks of follow-up was increased in male and decreased in female offspring. Both sexes showed increased liver weights, hepatic foci of cellular alterations and nuclear dysmorphology. In females, reductions in perigonadal and perirenal fat pad weights, serum triglycerides and cholesterol were also observed. Endocrine parameters, such as glucose tolerance, serum insulin and leptin, were not affected. In conclusion, our study with perinatal exposure to PFOA in mice produced metabolic effects in adult offspring. This is most likely due to disrupted programming of metabolic homeostasis, but the assayed endpoints did not provide a mechanistic explanation. The BMDL of the programming effects in our study is below the current point of departure used for calculation of the tolerable daily intake.

Compound- and sex-specific effects on programming of energy and immune homeostasis in adult C57BL/6JxFVB mice after perinatal TCDD and PCB 153.

Early life exposure to endocrine disrupting compounds has been linked to chronic diseases later in life, like obesity and related metabolic disorders. We exposed C57BL/6JxFVB hybrid mice to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the constitutive androstane receptor/pregnane X receptor agonist polychlorinated biphenyl 153 (PCB 153) in an experimental design relevant for human exposure. Exposure occurred during gestation and lactation via maternal feed to a wide dose range (TCDD: 10-10,000 pg/kg body weight/day; PCB 153: 0.09-1406 µg/kg body weight/d). Then exposure was ceased and offspring were followed up to 1 year of age. Metabolic parameters like body weight, fat pad weights, glucose tolerance, endocrine serum profile, and neurobehavioral and immunological parameters were determined. Body weight was transiently affected by both compounds throughout the follow-up. TCDD-exposed males showed decreased fat pad and spleen weights and an increase in IL-4 production of splenic immune cells. In contrast, females showed increased fat pad weights and production of IFNγ. PCB 153-exposed males showed an increase in glucose, whereas females showed an increase in glucagon, a decrease in pancreas weight, and an increase in thymus weight. In conclusion, early life exposure to TCDD appears to affect programming of energy and immune homeostasis in offspring, whereas the effects of perinatal PCB 153 were mainly on programming of glucose homeostasis. Both compounds act sex-specifically. Lowest derived BMDLs (lower bounds of the (two sided) 90%-confidence interval for the benchmark dose) for both compounds are not lower than current tolerable daily intakes.

Liver DNA methylation analysis in adult female C57BL/6JxFVB mice following perinatal exposure to bisphenol A.

Bisphenol A (BPA) is a compound released from plastics and other consumer products used in everyday life. BPA exposure early in fetal development is proposed to contribute to programming of chronic diseases like obesity and diabetes, by affecting DNA methylation levels. Previously, we showed that in utero and lactational exposure of C57BL/6JxFVB hybrid mice via maternal feed using a dose range of 0-3000µg/kg body weight/day resulted in a sex-dependent altered metabolic phenotype in offspring at 23 weeks of age. The most univocal effects were observed in females, with reduced body weights and related metabolic effects associated with perinatal BPA exposure. To identify whether the effects of BPA in females are associated with changes in DNA methylation, this was analyzed in liver, which is important in energy homeostasis. Measurement of global DNA methylation did not show any changes. Genome-wide DNA methylation analysis at specific CpG sites in control and 3000µg/kg body weight/day females with the digital restriction enzyme analysis of methylation (DREAM) assay revealed potential differences, that could, however, not be confirmed by bisulfite pyrosequencing. Overall, we demonstrated that the observed altered metabolic phenotype in female offspring after maternal exposure to BPA was not detectably associated with liver DNA methylation changes. Still, other tissues may be more informative.

Programming of metabolic effects in C57BL/6JxFVB mice by exposure to bisphenol A during gestation and lactation.

The global rise in prevalence of obesity is not fully explained by genetics or life style factors. The developmental origins of health and disease paradigm suggests that environmental factors during early life could play a role. In this perspective, perinatal exposure to bisphenol A (BPA) has been indicated as a programming factor for obesity and related metabolic disorders later in life. Here we study early life programming by BPA using an experimental design that is relevant for human exposure. C57BL/6JxFVB hybrid mice were exposed during gestation and lactation via maternal feed to 8 non-toxic doses (0-3000 µg/kg body weight/day (µg/kg bw/d)) of BPA. After weaning, offspring were followed for 20 weeks without further exposure. Adult male offspring showed dose-dependent increases of body and liver weights, no effects on fat pad weights and a dose-dependent decrease in circulating glucagon. Female offspring showed a dose-dependent decrease in body weight, liver, muscle and fat pad weights, adipocyte size, serum lipids, serum leptin and adiponectin. Physical activity was decreased in exposed males and suggested to be increased in exposed females. Brown adipose tissue showed slightly increased lipid accumulation in males and lipid depletion in females, and ucp1 expression was dose-dependently increased in females. The effects in females were more reliable and robust than in males due to wide confidence intervals and potential confounding by litter size for male data. The lowest derived BMDL (lower bound of the (two-sided) 90%-confidence interval for the benchmark dose) of 233 µg/kg bw/d (for interscapular weight in females) was below the proposed BMDL of 3633 µg/kg bw/d as a basis for tolerable daily intake. Although these results suggest that BPA can program for an altered metabolic phenotype, the sexual dimorphism of effects and diversity of outcomes among studies similar in design as the present study do not mark BPA as a specific obesogen. The consistency within the complex of observed metabolic effects suggests that upstream key element(s) in energy homeostasis are modified. Sex-dependent factors contribute to the final phenotypic outcome.

Toxicity of tetrabromobisphenol A (TBBPA) in zebrafish (Danio rerio) in a partial life-cycle test.

Toxicological effects of the widely used flame retardant, tetrabromobisphenol A (TBBPA) were assessed in a partial life-cycle test with zebrafish (Danio rerio). Exposure of adult fish during 30 days to water-borne TBBPA in nominal concentrations ranging from 0 (control) to 1.5 microM was followed by exposure of the offspring in early life stages up to 47 days posthatching (dph) to the same concentrations. Adults exposed to 3 and 6 microM showed severe disorientation and lethargy shortly after beginning of exposure and were euthanized. Because semistatic exposure resulted in fluctuating water concentrations, pooled fish samples were chemically analyzed for internal dose assessment. Egg production was decreased in fish exposed to TBBPA concentrations of 0.047 microM and higher, and a critical effect level of 7.2 microg/g lipid with a lower 5% confidence limit of 3.9 microg/g lipid for 50% decreased egg production was calculated. Histology of adult ovaries indicated a relative increase of premature oocytes in two surviving females exposed to 1.5 microM. Hatching of TBBPA-exposed larvae was decreased except in animals exposed to 0.375 microM. In the highest exposure concentration, early posthatching mortality was high (81%) in larvae and the surviving juveniles showed a significant predominance of the female phenotype. Exposure of eggs from control parents up to 6 microM TBBPA resulted in increasing malformation and pericardial fluid accumulation from 1.5 microM; at higher concentrations, all embryos failed to hatch. The presented results indicate decreased reproductive success in zebrafish at environmentally relevant TBBPA concentrations.

Aquatic toxicology: opportunities for enhancement through histopathology.

This paper briefly reviews the application of histopathology as aninstrument or endpoint in toxicity studies in fish. For long this has been applied rather occasionally in (regulatory) toxicology, and was mainly of interest in fundamental studies and limited carcinogenicity experiments. However, nowadays there are various incentives that ask for the application of pathology, such as field monitoring of pollution effects, the wish for optimal use and lower species of laboratory animals, the availability of modern histology techniques, and insight and interest in mechanistic data. This is timely illustrated by the current broad interest in endocrine disrupting pollutants-a threat mainly in the aquatic environment-where histopathological organ and tissue changes in intact sentinel fish species provide pivotal diagnostic and mechanistic features.

A new rat model of otitis media caused by Streptococcus pneumoniae: conditions and application in immunization protocols.

Streptococcus pneumoniae (pneumococcus [Pn]) can be cultured from up to 50% of acute otitis media (AOM) effusions, and these bacteria are the most common cause of AOM-related complications. With the recent advent of antibiotic-resistant Pn strains, treatment of Pn infections may meet with serious difficulties. Prevention through vaccination, notably for the four most common occurring Pn serotypes in humans (i.e., Pn 6B, Pn 14, Pn 19F, and Pn 23F), is a helpful alternative. Testing of vaccine efficacy should occur in an appropriate animal AOM model, which is presented here. The four involved Pn serotypes are not pathogenic to the rat, which was chosen as the experimental animal for practical reasons. To induce a natural infection (i.e., ascending through the eustachian tube), the mucociliary clearance of the eustachian tube was impaired by infusing histamine into the tympanic cavity on 2 consecutive days before intranasal inoculation of the bacteria. With this simple protocol, high and reproducible infection rates, as determined with bacterial cultures, of Pn-induced AOM (approximately 70%) with the two major Pn serotypes 14 and 19F (Pn 14 and Pn 19F) were obtained, whereas lower infection rates (25 to 50%) with Pn 6B and Pn 23F were obtained. In this model, intranasal priming with pneumococci, as well as subcutaneous vaccination with Pn 14 tetanus toxoid-conjugated polysaccharide, induced a protective effect against the induction of otitis media with these bacteria. This shows that immunity to Pn 14 AOM can be induced by both mucosal and systemic presentations of antigen. In conclusion, we have developed an animal model for Pn-induced AOM, which is suitable for the evaluation of the protecting effect of immunization.

Expression of insulin-like growth factor II (IGF-II) and histological changes in the thymus and spleen of transgenic mice overexpressing IGF-II.

Previously, transgenic mice were constructed overexpressing human insulin-like growth factor II (IGF-II) under control of the H2kb promoter. The IGF-II transgene was highly expressed in thymus and spleen, and these organs showed an increase in weight. In the current study we have analyzed the sites of IGF-II mRNA expression, the distribution of IGF-II, IGF-I, and both IGF receptors, and histomorphometrical changes in thymus and spleen. With in situ mRNA hybridization, expression of the IGF-II transgene is found with high intensity in the thymic medulla and in the white pulp/marginal zone of the spleen, whereas there were scattered positive cells in the thymic cortex and in the splenic red pulp. Hybridization was restricted to non-lymphocytic cells. Immunohistochemistry revealed intense IGF-II peptide staining with the same distribution as IGF-II mRNA. There was additional intense IGF-II staining of all elements in the splenic red pulp (including trabeculae) and diffuse, low level staining in the thymic cortex. These findings were not observed in control mice. In the thymic medulla, most IGF-II producing cells co-labelled with keratin, whereas a minor population also stained for the monocyte/ macrophage marker MOMA-2. In the spleen, co-labelling of IGF-II producing cells was found with MOMA-1 (marginal zone), or with the dendritic cell marker NLDC-145 (red pulp). IGF-I and both IGF receptors were found in these organs in nearly all cell types, with a similar pattern in transgenic mice and in control animals. Histomorphometric analysis revealed a marked increase of thymus cortex size and an increased trabecular size in the spleen. This suggests that IGF-II overproduction induces local effects (auto/paracrine) in the thymic cortex, but not in the thymic medulla. Trabecular growth in the spleen most likely is a distant effect (paracrine or endocrine) of IGF-II overproduction.

Expression of insulin-like growth factors (IGFs), their receptors and IGF binding protein-3 in normal, benign and malignant smooth muscle tissues.

To assess the role of insulin-like growth factors (IGFs) in growth and transformation of normal (myometrium) and tumorous smooth muscle cell (SMC) tissues, in situ hybridization (ISH) analysis for insulin-like growth factor I and II (IGF-I and IGF-II) mRNAs was combined with detection of IGF peptides, their receptors and IGF binding protein-3 (IGFBP-3). mRNAs for both IGFs were detected in smooth muscle cells in normal, benign and malignant SMC tissues, together with the IGF peptides, both IGF receptors and IGFBP-3. This suggests an autocrine role for both IGFs. Leiomyomas had higher IGF-I peptide levels and higher levels of type I IGF receptors than myometrium, supporting the idea that IGFs play a role in the growth and transformation of these tumours. Low-grade leiomyosarcomas contained more IGF-II mRNAs than myometrium and leiomyoma, fewer type II IGF/mannose 6-phosphate receptors and less IGFBP-3 than myometrium and, in addition, fewer IGF-I mRNAs and type I IGF receptors than leiomyoma. Intermediate- and high-grade leiomyosarcomas had intermediate levels of IGF-II mRNAs and peptide, ranging between those in myometrium and low-grade leiomyosarcomas. Thus, growth and transformation of leiomyosarcomas may be regulated by IGF-II, although more markedly in low-grade than in high-grade leiomyosarcomas. In conclusion, the various categories of SMC tissues are associated with a distinct expression pattern of the IGF system. This suggests that each category of SMC tumours arises as a distinct entity and that there is no progression of transformation in these tissues.

Histamine-stimulated expression of insulin-like growth factors in human glioma cells.

Glioma tumour growth is associated with the expression of insulin-like growth factors I and II (IGFs) and of both type I and type II IGF receptors. It has also been shown that IGFs can stimulate proliferation of cultured glioma cells. We previously reported that histamine too can stimulate the growth of glioma cells in vitro. In this report, we study whether the histamine-induced growth of G47 glioma cells is mediated by the IGFs. We found that histamine stimulates the expression of both IGF-I and IGF-II mRNAs, as determined by a semiquantitative in situ hybridization analysis. Furthermore, incubation of G47 cells with histamine also induced cellular immunostaining for IGF-II. It could be shown that IGF-I-stimulated proliferation is inhibited by IGFBP-3, which decreases the availability of IGFs for binding to the IGF receptors, and by beta-galactosidase, which may decrease IGF binding to the type II IGF receptor, but is not inhibited by the anti-type I IGF receptor monoclonal antibody alphaIR3. However, neither IGFBP-3 nor beta-galactosidase nor alphaIR3 inhibited the histamine-induced proliferation. These results show that the growth-stimulatory effect of histamine is accompanied by the induction of IGFs. This histamine-induced growth stimulation is not mediated by activation of cell surface IGF receptors, although intracrine activation of type II IGF receptors may be involved.

Modulation of insulin-like growth factor (IGF) action by IGF-binding proteins in normal, benign, and malignant smooth muscle tissues.

The insulin-like growth factor (IGF) system is involved in the growth of uterine leiomyomas (L), as these tumors have higher IGF-II messenger ribonucleic acid levels, type I IGF receptor levels, and IGF-I peptide concentrations than myometrium (M). Furthermore, cultured L smooth muscle cells (SMC) respond with greater efficiency to IGF-I than M SMC. Here we investigate a possible modulating role of the binding proteins for the IGFs (IGFBPs) on the actions of IGFs. IGFBP-3 is the most predominant IGFBP in conditioned medium from SMC, with levels ranging from 13-288 ng/mL. Incubation of SMC cultures with IGF-I and the IGF-I analogs long-R3IGF-I and des(1-3)-IGF-I, which have decreased affinity for IGFBPs, revealed a facilitating effect of IGFBPs on the growth-stimulating activity of a high concentration of IGF-I in cell lines with high IGFBP-3 levels. Both a decreased level of IGFBP-3 and a low concentration of the growth factors added were a disadvantage for the facilitating effect. In M and L tissue sections, IGFBP-3 was found exclusively bound to the constituting cells, not in the extracellular matrix. This suggests that a negative modulating role of IGFBP-3 due to sequestration of IGF-I, as occurs in culture medium, is less relevant in vivo. In leiomyosarcoma sections, IGFBP-3 levels are decreased, indicating a decreasing, role for this binding protein in malignant smooth muscle tissues.

Ocular squamous cell carcinoma in Simmental cattle in Zimbabwe.

In Zimbabwe, ocular squamous cell carcinoma (OSCC) was frequently observed in 5 breeding herds of Simmental cattle, a Bos taurus breed originating from Switzerland. In these herds, initial signs of OSCC were already noticeable in cattle about 3 years old. Gradually, OSCC prevalence increased, and 36 to 53% of cattle over 7 years old had 1 or more tumors. More tumors developed in Simmental cattle with periorbital white skin than in cattle with periorbital pigmented skin. Other breeds of cattle (eg, Friesian) also are partly white-faced and live in Zimbabwe in a comparable environment; yet, OSCC prevalence was lower in those breeds.

Growth advantage of human leiomyoma cells compared to normal smooth-muscle cells due to enhanced sensitivity toward insulin-like growth factor I.

Human uterine leiomyomas exhibit increased IGF-I binding compared to myometrium, while both tissues show IGF-I gene expression. In this study we have examined the functional importance of these findings by testing the presence of IGF-I in 15 leiomyoma biopsies and in 18 myometrium biopsies and the capacity of smooth-muscle cells cultured from these tissues to react to IGF-I. The mean IGF-I peptide concentration in leiomyomas was 3 times higher than in myometrium. This resulted from increased IGF-I uptake in leiomyomas rather than from increased synthesis, as these tissues contain higher concentrations of type-I IGF receptors, as detected by immunohistochemistry, and equal levels of IGF-I mRNA. Blocking IGF-I transport with cytochalasin-B and with the type-I IGF receptor blocking antibody alpha IR3 in cultured cells induced decreased immunostaining intensity for IGF-I in most myometrium and leiomyoma cultures, indicating that the detected IGF-I is internalized. Depending on the culture conditions, IGF-I administration yielded increased survival or a higher proliferation rate in leiomyoma cultures than in myometrium cultures, indicating the increased importance of exogenous IGF-I for the growth of transformed smooth-muscle cells. We conclude that the increased concentrations of type-I IGF receptors in leiomyoma compared to myometrial smooth-muscle cells are functional with respect to the enhanced internalization of IGF-I and that they provide these tumor cells with a growth advantage compared to their normal counterparts.

31P NMR phospholipid characterization of intracranial tumors.

Phospholipid extracts from 48 intracranial tumors were analyzed using 31P NMR. Phospholipids commonly identified in the tumor spectra included phosphatidylglycerol (PG), phosphatidic acid (PA), diphosphatidylglycerol (DPG), uncharacterized phospholipid (U), ethanolamine plasmalogen (EPLAS), phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (SM), lysophosphatidylcholine (LPC), phosphatidylinositol (PI), a choline phospholipid (CPLIP), and phosphatidylcholine (PC). Differences in the mean relative mole-percentage of phosphorus concentrations of individual phospholipids were used to differentiate among tumors. Neural sheath tumors (neurilemmoma, neurofibroma and fibrosarcoma) were noted to contain significantly elevated levels of SM relative to tumors of neural glial origin and individually, glioblastoma multiforme was noted to contain depressed levels of SM relative to neurilemmoma, neurofibroma and meningioma. Significantly decreased levels of PA were noted for glioblastoma relative to neurilemmoma along with significantly decreased levels of PE relative to meningioma. Elevated levels of LPC and CPLIP were seen in glioblastoma multiforme relative to meningioma. Additional findings included elevated levels of PC for glioblastoma multiforme relative to neurofibroma, and neurilemmoma was differentiated from neurofibroma with elevated levels of PA and depressed levels of PI. 31P NMR phospholipid analysis provides supplemental biochemical information which may be used to improve the interpretation of spectra acquired in vivo, and reveals important tumor-specific biochemical information which may further improve the understanding of the biological behavior of intracranial tumors.

Meningioma phospholipid profiles measured by 31P nuclear magnetic resonance spectroscopy.

Fourteen cases of intracranial meningioma were characterized after chloroform/methanol extraction by 31P nuclear magnetic resonance (NMR) spectroscopy at 202.4 MHz. Each phospholipid class detected in the extracts was identified and quantitated in terms of its molar percentage relative to the total phospholipids measured. The following phospholipids were assayed by 31P NMR: phosphatidylglycerol, phosphatidic acid, diphosphatidylglycerol, ethanolamine plasmalogen, phosphatidylethanolamine (PE), lysophosphatidylinositol, phosphatidylserine, sphingomyelin, lysophosphatidylcholine (LPC), phosphatidylinositol (PI), sphingosylphosphorylcholine and phosphatidylcholine. In addition, two unidentified phospholipids were detected with resonances at 0.13 and -0.78 ppm, respectively. Three distinct types of spectra were obtained on the extracts and grouped accordingly for comparison purposes. Type 1 tumors showed unusual 31P NMR profiles with low levels of PE and PI and elevated levels of LPC; type 2 tumors were characterized by low levels of the ethanolamine phospholipids and near equivalent levels of PI and LPC. The spectra of type 1 and type 2 tumors were characteristic of degenerative cells that lacked membrane permeability associated with loss of ethanolamine plasmalogen in the presence of significant phospholipid turnover. Meningiomas belonging to the third spectral type showed characteristics similar to those of normal tissues with normal levels of PE and ethanolamine plasmalogen, as well as very low levels of LPC relative to PI. Type 3 tumors lacked the characteristic signs of degeneration noted in type 1 and type 2 tumors. The data corroborate and augment in vivo spectroscopic findings reported earlier and demonstrate the value of 31P NMR spectroscopic phospholipid analysis on lipid extracts for the characterization of meningiomas.

An avaII restriction fragment length polymorphism in the insulin-like growth factor II gene and the occurrence of smooth muscle tumors.

To detect a previously described AvaII restriction fragment length polymorphism (RFLP) in the human insulin-like growth factor II (IGF-II) gene we used the polymerase chain reaction (PCR) and genomic sequencing. The RFLP is located in exon 9 of the IGF-II gene at nucleotide 820 (GenBank accession number X07868) as a C-->T transition. Digestion with AvaII reveals a two-allele polymorphism, an a allele in which the AvaII site is not present, and a b allele. In healthy Dutch persons (n = 26), the frequency of the a allele was 62%. A similar a allele frequency was found in groups of Japanese (53%, n = 65) and Chinese (54%, n = 84), while in a French group the frequency was significantly lower (25%, n = 52). In Dutch individuals that had developed benign (n = 11; all women) and malignant (n = 9; 2 women and 7 men) smooth muscle tumors, a significantly higher frequency of 83% for the a allele was found. Since there was no difference between the presence of the a and b alleles in normal and tumor tissue of the same individual, the higher a allele frequency was not due to mutation in the IGF-II gene or loss of heterozygosity. There was no correlation between the presence of the a allele and expression of the IGF-II gene. The data reveal a correlation between homozygosity for the a allele and the occurrence of smooth muscle tumors. Women homozygous for the IGF-II a allele are more prone to develop a leiomyoma than women who are heterozygous or homozygous for the b allele. Furthermore, in both women and men the risk for leiomyosarcomas seems to be higher in a allele homozygotes.

Growth of cultured human glioma tumour cells can be regulated with histamine and histamine antagonists.

The 50% survival time for low grade astrocytomas is 50 months and for high grade astrocytomas it is 13 months, underlining the need for new therapies. Several reports show that in vivo histamine antagonists cause retardation of tumour growth in some animal models and prolonged survival in cancer patients. Therefore we have tested the growth modulating effects of histamine and histamine antagonists on human glioma cultures. Twelve freshly excised human gliomas were cultured and tested for their in vitro sensitivity to histamine and histamine antagonists. Four continuous glioma cell lines were used to confirm the glioma-specificity of the effects observed in the primary cell lines. In low serum concentration (0 or 1%) the growth of 5/9 primary glioma-derived cultures could be stimulated with 0.2 mM histamine, and in 4/5 cases with 0.2 microM histamine. One mM of the histamine H2-receptor antagonist cimetidine could inhibit the growth of 4/5 primary glioma cultures when tested in 1% human AB serum, and of 6/13 cases when tested in 1% FCS. Lower concentrations (down to 1 microM) were less effective. The histamine H1-receptor antagonist pyrilamine gave variable results. The specificity of the effects is indicated by the absence of a generalised toxic effect, by the observation that the antagonist-induced inhibition could be reversed with histamine, and by the correlation of the obtained cimetidine-induced growth inhibition with the maximal growth rate of the primary cell lines in 10% FCS. The observed cimetidine-induced inhibition of the in vitro proliferation of gliomas suggests that cimetidine is a relevant candidate for the in vivo growth inhibition of these tumours.

Expression and CpG methylation of the insulin-like growth factor II gene in human smooth muscle tumors.

Previously we have shown that expression of the insulin-like growth factor II (IGF-II) gene in 36 normal smooth muscle tissues (myometria) and 26 benign smooth muscle tumors (leiomyomas) was detectable by Northern blot analysis but that the RNA levels were low. In 9 of 20 malignant smooth muscle tumors (leiomyosarcomas) IGF-II gene expression was also low or absent, while in 11 of 20 the IGF-II gene was abundantly expressed. In 32 of these tissues we have now studied the DNA methylation state of the IGF-II gene. For the analysis of overall methylation of the gene the restriction endonucleases HpaII and MspI were used. In normal smooth muscle and in leiomyomas the IGF-II gene appeared to be methylated. In leiomyosarcomas with low IGF-II gene expression the DNA was partly demethylated. In leiomyosarcomas with abundant IGF-II gene expression overall methylation of the DNA tended to be low. In addition, we have studied the methylation state of one particular CpG site in the IGF-II gene with the restriction endonuclease AvaII. The results of the latter analysis confirm the analysis with HpaII and MspI. In conclusion, in malignant smooth muscle tumors the data indicate an inverse correlation between CpG methylation and expression of the IGF-II gene.