Endogenous opioids and reward.

The discovery of endogenous opioids has markedly influenced the research on the biology of addiction and reward brain processes. Evidence has been presented that these brain substances modulate brain stimulation reward, self-administration of different drugs of abuse, sexual behaviour and social behaviour. There appears to be two different domains in which endogenous opioids, present in separate and distinct brain regions, are involved. One is related to the modulation of incentive motivational processes and the other to the performance of certain behaviours. It is concluded that endogenous opioids may play a role in the vulnerability to certain diseases, such as addiction and autism, but also when the disease is present, such as alcoholism.

Morphine attenuates the effects of juvenile isolation in rats.

The acute effects of juvenile isolation on sucrose intake and its long-term consequences on adult social behavior were investigated. Additionally, the role of the endogenous opioid systems was studied. Juvenile rats were housed in one of three conditions: in groups or in isolation with (partial isolation, PI) or without 30 min of daily social contact from 22 to 35 days-of-age. During this period the rats were treated daily with saline or morphine. Juvenile isolated rats showed an increased sucrose intake as compared to non-isolated controls, with PI-rats somewhere in-between, suggesting a negative correlation between the amount of social contact and sucrose consumption. Morphine treatment during the isolation period enhanced the sucrose intake in non-isolated rats, whereas it decreased sucrose consumption in (partial) isolated rats. With regard to the long-term effects, (partial) isolated rats decreased social activity as compared to non-isolated controls which was reversed by morphine treatment during the isolation period. In non-isolated rats, morphine treatment caused an opposite effect: it decreased social activity as compared to the saline treated controls. The data suggest that stimulation of endogenous opioid systems in the juvenile phase may have an important modulatory role in the expression of adult social behavior. The results are discussed in relation to a possible function of morphine as a substitute for the release of endogenous opioid peptides during social play.

Isolation changes the incentive value of sucrose and social behaviour in juvenile and adult rats.

The present study was undertaken to assess the motivational aspects of social behaviour in juvenile and adult rats using the conditioned place preference (CPP) test and anticipatory behaviour for social contact. In addition, the consequences of social isolation during different periods of age on the motivational properties of sucrose-drinking and adult social behaviour were studied. Social play and adult social contact could be used as incentives for place preference conditioning and for inducing conditioned hyperactivity (anticipation) in rats. Both social activities have motivational properties for individually housed rats, whereas group-housing dramatically reduced the motivational aspects of adult social contact. In contrast, sucrose-drinking appears to have motivational aspects independent of the housing condition. Adult social behaviour could not induce a CPP in juvenile isolated rats, suggesting that juvenile isolation during 4 5 weeks reduced the motivational aspects of adult social contact. It seems likely that no CPP was established as a result of the reduced level of social behaviour during the conditioning sessions. Additionally, juvenile isolation during 4-5 weeks appeared to also decrease the motivational properties of sucrose-drinking in maturity, because the intensity of anticipation in response to sucrose was significantly suppressed. Thus, the data suggest that juvenile isolation during 4-5 weeks decreases the motivational properties of both social contact and sucrose-drinking in later life.

Sequential analysis of juvenile isolation-induced decreased social behavior in the adult rat.

The consequences of juvenile isolation on adult social behavior were studied in detail using two different analysis methods: frequency, duration, and latency of behavioral elements, and sequential analysis. Rats were either isolated or socially housed during weeks 4 and 5 of age, and after the isolation period housed in pairs with a rat of identical housing condition until the time of testing at 12 weeks of age. Juvenile isolation caused marked effects on the frequency, duration, and latency of various social behavioral elements, whereas the non-social activities such as ambulation, rearing, and self-grooming were hardly affected. Juvenile isolation reduced social exploration, anogenital sniffing, and approach/following and increased the latency to the first occurrence of these social behavioral elements. In contrast, the sequential analysis revealed that the structure of social behavior was barely affected by juvenile isolation. Some transitions were less pronounced in juvenile isolated rats compared to non-isolated rats, but no significant differences were observed in transitions between social elements. Thus, juvenile isolation bisected the time spent on adult social interactions, whereas it did not disrupt the sequential structure of social behavior. The present data suggest that juvenile isolation reduced the motivation for adult social behavior, but when social contact is initiated, a relatively normal social behavioral pattern is displayed.

Effects of juvenile isolation and morphine treatment on social interactions and opioid receptors in adult rats: behavioural and autoradiographic studies.

The consequences of juvenile isolation and morphine treatment during the isolation period on (social) behaviour and mu-, delta- and kappa-opioid receptors in adulthood were investigated by using a social interaction test and in vitro autoradiography in rats. Juvenile isolation reduced social exploration in adults. Morphine treatment counteracted this reduction in isolated rats, but decreased social exploration in nonisolated rats. Self-grooming and nonsocial exploration were enhanced after juvenile isolation. Morphine treatment had no effect on self-grooming, but suppressed nonsocial exploration in isolated rats. With respect to the opioid receptors, juvenile isolation resulted in regiospecific increases in mu-binding sites with a 58% increase in the basolateral amygdala and a 33% increase in the bed nucleus of stria terminalis. Morphine treatment in isolated rats reversed this upregulation in both areas. The number of delta-binding sites did not differ between the experimental groups. A general upregulation of kappa-binding sites was observed after juvenile isolation, predominantly in the cortical regions, the hippocampus and the substantia nigra. Morphine treatment did not affect the upregulation of kappa-receptors. The results show that juvenile isolation during the play period causes long-term effects on social and nonsocial behaviours and on the number of mu- and kappa- but not delta-opioid receptors in distinct brain areas. The number of mu-receptors in the basolateral amygdala appears to be negatively correlated with the amount of social exploration in adult rats.

Insulin-like growth factor (IGF)-I rescues breast cancer cells from chemotherapy-induced cell death--proliferative and anti-apoptotic effects.

Insulin-like growth factor (IGF)-I protects many cell types from apoptosis. As a result, it is possible that IGF-I-responsive cancer cells may be resistant to apoptosis-inducing chemotherapies. Therefore, we examined the effects of IGF-I on paclitaxel and doxorubicin-induced apoptosis in the IGF-I-responsive breast cancer cell line MCF-7. Both drugs caused DNA laddering in a dose-dependent fashion, and IGF-I reduced the formation of ladders. We next examined the effects of IGF-I and estradiol on cell survival following drug treatment in monolayer culture. IGF-I, but not estradiol, increased survival of MCF-7 cells in the presence of either drug. Cell cycle progression and counting of trypan-blue stained cells showed that IGF-I was inducing proliferation in paclitaxel-treated but not doxorubicin-treated cells. However, IGF-I decreased the fraction of apoptotic cells in doxorubicin- but not paclitaxel-treated cells. Recent work has shown that mitogen-activated protein kinase (MAPK) and phosphotidylinositol-3 (PI-3) kinase are activated by IGF-I in these cells. PI-3 kinase activation has been linked to anti-apoptotic functions while MAPK activation is associated with proliferation. We found that IGF-I rescue of doxorubicin-induced apoptosis required PI-3 kinase but not MAPK function, suggesting that IGF-I inhibited apoptosis. In contrast, IGF-I rescue of paclitaxel-induced apoptosis required both PI-3 kinase and MAPK, suggesting that IGF-I-mediated protection was due to enhancement of proliferation. Therefore, IGF-I attenuated the response of breast cancer cells to doxorubicin and paclitaxel by at least two mechanisms: induction of proliferation and inhibition of apoptosis. Thus, inhibition of IGF-I action could be a useful adjuvant to cytotoxic chemotherapy in breast cancer.

Morphine treatment during juvenile isolation increases social activity and opioid peptides release in the adult rat.

The consequences of juvenile isolation and morphine treatment on general activity, social activity and endogenous opioid release during a social interaction test were investigated in the adult rat. Rats were either isolated or socially housed during weeks 4 and 5 of age and treated daily during this isolation period subcutaneously with either saline or morphine. Directly after a social interaction test at 10 weeks of age, rats were injected with [3H]-diprenorphine and subsequently prepared for in vivo autoradiography. The autoradiographic technique was used to visualise neuroanatomical changes in opioid receptor occupancy, probably reflecting changes in opioid peptide release, as a result of social activity. Juvenile isolation increased general activity during the social interaction test, an effect which was accompanied by a reduction of opioid receptor occupancy in many brain areas, suggesting an increased opioid peptide release as a consequence of socially-induced general activity. Morphine treatment in isolated rats caused an increase in adult social activity and enhanced opioid peptide release in some cortical regions and the ventral tegmental area as compared to saline treated rats. Both social activity and opioid receptor occupancy were unaffected by morphine treatment in non-isolated rats. The present study underscores the role of opioid systems in adult social behaviors as a consequence of juvenile isolation. The results suggest a relationship between social activity and opioid peptide release during social contact. Increased social activity seems to be accompanied by elevated opioid peptide release in distinct brain areas after morphine treatment during juvenile isolation.

Isolation during the play period in infancy decreases adult social interactions in rats.

The effects of 1 or 2 weeks of social isolation immediately after weaning on social activity in adulthood were investigated in rats. In addition, it was studied whether these effects were influenced by social experiences of the cagemate when rehoused after the isolation period. Isolation during weeks 4 and 5 of age caused a reduction of social activity as compared to non-isolated controls. Previous social experiences of the cagemate (isolated or non-isolated) did not affect this decreased social activity. Isolation during week 4 of age resulted in similar effects, but the reduced social activity was not present when the rats were rehoused with non-isolated rats. Isolation during week 5 of age did not influence social activity patterns in adulthood. These findings support the idea of a sensitive period in infancy for subsequent social behavior in rats. It is suggested that especially deprivation of acquiring play behavior underlies the social disturbances in adulthood.

Play is indispensable for an adequate development of coping with social challenges in the rat.

In this study, young rats were deprived of early social interactions during weeks 4 and 5 of life. Different behavioral tests were conducted in adulthood to study the behavioral responses of rats lacking early social experiences. Juvenile deprivation resulted in decreased social activity and an altered sexual pattern, but did not affect locomotor activity or the performance in the elevated plus maze. Furthermore, behavioral and neuroendocrine responses of juvenile isolated rats were dramatically altered when they were confronted with territorial aggression. Juvenile deprived rats did not readily display a submissive posture in response to the resident and showed no immobility behavior after being returned to the resident's territory, while their plasma corticosterone and adrenaline concentrations were significantly increased compared to nonisolated controls. In contrast, behavioral responses in the shock prod test were not affected by previous isolation. The results suggest that early social experiences are vital for interactions with conspecifics later in life, i.e., aggression, sexual, and social interactions.

Emotional and footshock stimuli induce differential long-lasting behavioural effects in rats; involvement of opioids.

Rats were exposed to either a footshock stimulus (FS) or emotional stimulus (ES, forced perception of another rat receiving footshocks) during a daily 10-min session for 5 consecutive days. The consequences of FS and ES on their behavioural responsiveness were assessed at different post-stress intervals using a small open-field. FS induced a decrease in ambulation, rearing and sniffing and an increased immobility in the small open field. These effects were present in rats tested immediately after the last session and remained present for at least 15 days. In contrast, ES induced a transient decrease in ambulation and rearing immediately after the last session, but in the period from half an hour until at least 15 days after the stimulus experience, an increase in ambulation, rearing and sniffing was observed. Exposure to one footshock per session for 5 consecutive days or to 10 footshocks in a single session also resulted in a long-lasting reduction in ambulation and sniffing and an increase in immobility. The former regime did not influence the behavioural response of ES rats, but the latter resulted in an increase in ambulation, rearing and sniffing in ES rats. Naloxone (1 mg/kg s.c.) pretreatment antagonized the increased behavioural activity of the ES rats whereas the activity of control and FS animals was not affected, suggesting an involvement of endogenous opioid systems in the behavioural responses observed in ES rats. It is suggested that the behavioural responses of the ES and FS animals are regulated by different mechanisms.

Polyethylene glycol conjugated insulin-like growth factor binding protein-1 (IGFBP-1) inhibits growth of breast cancer in athymic mice.

Insulin-like growth factor (IGF) binding protein-1 (BP-1) inhibits IGF-mediated proliferation of some breast cancer cell lines in vitro. Here we examined whether recombinant human wild-type IGFBP-1 (WT-BP-1) and IGFBP-1 conjugated with polyethylene glycol (PEG-BP-1) could inhibit breast cancer growth. Three breast cancer cell lines were used: MCF-7, MDA-MB-231 and MDA-MB-435A (ascites model). The cells were grown in agar with or without the BP-1 conjugates to investigate their effect on colony formation. Both WT-BP-1 and PEG-BP-1 inhibited anchorage-independent growth (AIG) of MCF-7 and MDA-MB-435A cells. AIG of MDA-MB-231 cells was not inhibited by PEG-BP-1, whereas WT-BP-1 significantly stimulated colony number. We also tested both forms of BP-1 in xenograft tumour models. Two solid breast tumour models were studied using MCF-7 and MDA-MB-231 cell lines, and one ascites model using the MDA-MB-435A cell line. PEG-BP-1 inhibited malignant ascites formation in the MDA-MB-435A model. Conversely, PEG-BP-1 did not significantly inhibit MCF-7 xenograft growth. However, the MDA-MB-231 tumour growth curves were significantly different by a constant amount, suggesting that PEG-BP-1 treatment inhibited early tumour growth of this cell line. In contrast, WT-BP-1 was ineffective in the MDA-MB-231 tumours. These data show that anti-IGF strategies can be used to inhibit breast cancer cell growth. Since PEG-BP-1 inhibited the in vivo, but not in vitro, growth of MDA-MB-231, we speculate that PEG-BP-1 may block host IGF functions required for optimal tumorigenesis. Because PEG-BP-1 has a prolonged serum half-life compared to WT-BP-1, we conclude that improvements in BP-1 pharmacological properties enhanced its antitumour effects in vivo.

Relationships between the regioselectivity of the hydroxylation of C4-substituted 2-fluoroaniline derivatives and their toxic endpoints.

The in vitro and in vivo metabolic profiles of a series of C4-substituted 2-fluoroanilines were determined and compared to their capacity to induce methemoglobinemia and nephrotoxicity in male Wistar rats. Qualitative and quantitative relationships between the biotransformation and the toxic endpoint of the halogenated anilines were defined. The rate of in vitro N-hydroxylation of the aniline derivatives correlated with the capacity of the compounds to induce methemoglobinemia (r = 0.96). In the experiments on the nephrotoxicity, attention was focused on the relative importance of the C4- and C6-hydroxylated metabolites of the C4-substituted 2-fluoroanilines. In vivo, the formation of 4-aminophenol metabolites was demonstrated to vary in the opposite order as the formation of the 6-aminophenol metabolites. 1H-NMR urinalysis and characterization of a set of conventional biochemical urinary parameters revealed the occurrence of nephrotoxicity upon exposure to the aniline derivatives and were most consistent with damage at the proximal tubular site. Comparison of the extent of nephrotoxicity to the extent of formation of the 4-aminophenol and/or 6-aminophenol metabolites, respectively, indicates a predominant role for the C4-hydroxylation route, not the C6-hydroxylation route, in the induction of nephrotoxic effects. Thus, a qualitative relationship is observed for the extent of C4-hydroxylation of the aniline derivatives and the extent of their in vivo nephrotoxicity. In addition, comparison of the extent of 4-aminophenol formation and nephrotoxicity of both 2-fluoroaniline and 2,4-difluoroaniline pointed at a possible role for a bioactivation pathway through oxidative dehalogenation, resulting in direct formation of a 1,4-benzoquinoneimine as the primary metabolite in the case of 2,4-difluoroaniline. Altogether, it is concluded that a decrease in C4-hydroxylation in the series of aniline derivatives results in a metabolic switch to C6- and N-hydroxylation and, consequently, a shift in the type of toxic endpoint observed, i.e., from nephrotoxicity to methemoglobinemia.

Pharmacokinetic profile of recombination human insulin-like growth factor binding protein-1 in athymic mice.

Neutralization of insulin-like growth factor action by insulin-like growth factor binding protein-1 inhibits the in vitro growth of breast cancer cells. We performed this study to determine the pharmacokinetic profile of recombinant human IGFBP-1 (rhIGFBP-1) in athymic mice as a prelude to testing this protein in a human tumor xenograft model. After the subcutaneous injection of 1 mg, rhIGFBP-1 migrating at 29 kDa could be detected by ligand blotting and immunoblotting. Plasma concentrations of rhIGFBP-1 were quantified by immunoassay and demonstrated a half-life was 2.49 hours with the maximal concentration of 43.5 micrograms/mL occurring at 1 hour. The area under the concentration-time curve was 78.32 micrograms x hr/mL. Plasma clearance was 12.77 mL/hr and the mean residence time was 1.96 hours. rhIGFBP-1 was also detected in some tissues and was also cleared rapidly. These results show that high plasma and tissue levels of rhIGFBP-1 can be obtained after subcutaneous injection in athymic mice, however, the short half-life of the protein may limit its therapeutic use.

Pharmacokinetics of hydroxyurea in nude mice.

Extrachromosomal DNA is the predominant form of gene amplification in human tumors. Hydroxyurea (HU) concentrations of 100-150 microM have been promising in vitro for extrachromosomal DNA elimination. The study objective was to determine the HU dose-concentration relationship in nude mice with HU doses from 0 to 200 mg/kg. For HU t1/2 determination, mice were injected with HU 100 mg/kg. A plasma concentration of 159 microM was achieved and a t1/2 of 11.3 min determined. Based on these findings, In vivo elimination studies will require frequent administration of HU to maintain plasma concentrations from 100 to 150 microM.