[Psychological impact of the COVID-19 pandemic: Comparison between residents of metropolitan France and of Reunion Island]. / Impact psychologique de la pandémie de la COVID-19 : comparaison entre des Français métropolitains et de l'île de La Réunion.

OBJECTIVES: The COVID-19 pandemic raised a lot of anxiety around the world. France is composed of several overseas territories with major cultural differences but also with a different exposure to the COVID-19. Reunion Island is the most populated overseas French department, but few researches have focused on this population. Therefore, the main objective was to explore and compare the impact of the COVID-19 pandemic (perceived stress, risk and fear of being infected, severity, lockdown respect, perceived stress, quality of life, quality of relationship, loneliness, resilience) during the lockdown among residents of metropolitan France and of Reunion Island. MATERIAL AND METHODS: A sample of 347 participants, aged from 18 to 78 (M=37.90; DS=13.20) replied to a questionnaire posted online during the last ten days of the lockdown in France. The sample is divided into 227 metropolitans (M=38.24 DS=13.41; 13.2% of men) and 120 residents of Reunion Island (M=37.26; DS=12.81; 31.7% of men). Resilience, loneliness and perceived stress have been assessed using validating scales while specific items have been created to assess COVID-19 impacts. RESULTS: The majority of the total sample has been little exposed to the COVID-19, but the estimated severity was high throughout the sample. Several significant differences have been observed between overseas and metropolitans. The latter, who were more exposed, were more respectful of lockdown measures and felt more concerned about being contaminated. They also had different professional activities (work at home, stop working) since the lockdown than did the overseas sample. Non-significant higher scores of resilience and quality of life during the lockdown contrast in the overseas sample, who estimated risk, fear and severity similarly. In correlational analyses, many relationships were significant only in one sample. For example, in metropolitans the higher the loneliness, the higher the severity. In the other sample, the higher the perceived stress, the higher the respect of lockdown measures, while more metropolitans felt lonely the more they respected these measures. Regressional analysis showed different predictive variables of the scores of perceived stress and fear of being contaminated. In metropolitans, stress was explained by COVID-19 related variables (fear, severity, respect), loneliness and negatively by resilience and quality of life while in the other sample it was explained by fear of being infected and negatively by resilience. Fear of being infected was explained by risk and stress in the overseas, but also by quality of life in the metropolitan sample. CONCLUSIONS: This study brings new data on the important psychosocial impact of the COVID-19 pandemic on two French samples. Observed differences highlight a higher fear of being infected among the metropolitans who were generally more exposed. Overseas from La Réunion did not feel more spared by this risk, despite the limited number of cases since the appearance of the first case in March 11th and the end of the lockdown in May 11th. Despite exposure, our results could be explained by several cultural differences such as way of life or beliefs. Overseas life in Reunion Island might bring more resilience and less loneliness given the particular familial, social and religious functioning. Given the limits of this study and the lack of similar comparisons, more work could highlight the protective factors of these populations.

Need for systematic screening for IGD in adolescent psychiatric inpatients.

Psychiatric comorbidities are frequent in adolescents with internet gaming disorder (IGD). In contrast, the proportion of IGD among adolescents hospitalized for a psychiatric disorder has not been documented yet. In addition, parental ratings of IGD could be useful for diagnosis, but very few data exist on this issue. The objectives of this study were to: (1) assess the prevalence of IGD among adolescent psychiatric inpatients, using the Ten-Item Internet Gaming Disorder Test (IGDT-10), and (2) assess the parental version developed for this study (IGDT-10-P). A total of 102 patients, aged from 12 to 17 years old, were included from four psychiatric units of the French region Auvergne-Rhône-Alpes, during a 6-month inclusion period. Adolescents completed the IGDT-10 while one of their parents completed the IGDT-10-P. The inclusion rate among the eligible population was 57.95%. The prevalence of IGD in the sample, based on the IGDT-10 and IGDT-10-P, was 6.00% and 12.79%, respectively. Psychometric features of the IGDT-10-P indicated excellent internal consistency, a good model fit to the one factor model in confirmatory factor analysis, a strong correlation with gaming time, and a moderate correlation with the IGDT-10. Our results support the need for a systematic screening of IGD among adolescents hospitalized for a psychiatric disorder. Future studies should aim to confirm and explain the prevalence gap between self- and parent-reported criteria.

The Generalized Problematic Internet Use Scale-2 in a French sample: Psychometric evaluation of the theoretical model.

OBJECTIVES: The Generalized Problematic Internet Use Scale-2 (GPIUS-2) is a short self-report questionnaire assessing Internet addiction based on a cognitive behavioral model. Our main goal was to evaluate the psychometric properties of its French version among a sample of students and to appraise the relevance of the generalized problematic Internet use model. METHODS: A sample of 563 university students aged between 18 and 35 years (M=20.8; SD=2.7) completed several online self-report questionnaires including the GPIUS-2, the Internet Addiction Test (IAT) and the Center for Epidemiologic Study-Depression scale (CES-D). RESULTS: Confirmatory Factorial Analyses revealed a poor but acceptable overall fit for the original five-factor model and the original four-factor model. Path analyses, testing Structural Equation Modeling provided showed a poor fit to the data, suggesting insufficient construct validity. Convergent and concurrent validities analyzed through correlational analyses revealed significant relationships between the GPIUS-2, its factors, the IAT, time spent online and the CES-D. CONCLUSIONS: This research highlights the insufficient psychometric properties of the GPIUS-2 in a French sample, similar to previous results. However, this French version appears to be a useful multidimensional tool for assessing problematic Internet use among students, and reveals promise for future research and clinical applications of the measure, given its solid theoretical basis and despite the results of this psychometric study.

[Problematic Internet use, time spent online and personality traits]. / Utilisation problématique d'Internet, temps passé en ligne et traits de personnalité.

BACKGROUND: Internet addiction or problematic Internet use is a recent and increasingly recognized disorder which has been consistently associated with many psychiatric disorders, adding to the documented negative consequences of problematic Internet use. However, very few studies have examined the relationship between problematic Internet use and personality traits and none in a French sample. Moreover, those which have evaluated this relationship have mainly been conducted on small samples. OBJECTIVE: The main goal of our study was to explore the relationship between problematic Internet use, time spent online and personality traits in a French sample, taking into account the presence of depressive symptoms, and gender. METHODS: A sample of 276 participants aged from 18 to 50 (M=28; SD=8.9) completed a questionnaire assessing problematic Internet use, time spent online, the presence of ten personality traits and depressive symptoms. RESULTS: Our results revealed significant differences between genders. Among men, problematic Internet use was associated with personality clusters A and B while in women no cluster or personality traits were associated. Time spent online was predicted by schizoid personality traits among men and avoidant personality traits among women. DISCUSSION: Our results indicate that cluster A (schizoid and schizotypal) and cluster B traits (borderline and antisocial) play a more important role in problematic Internet use than cluster C traits among men. Differences between men and women regarding the relationships between personality traits, time online and problematic Internet use may be related to differences in the activities engaged in by men and women online. We observed that communication websites use was more prevalent among women while erotic, gambling and shopping websites use was more prevalent among men suggesting that the characteristics of problematic Internet use may vary according to gender. CONCLUSION: Few studies have examined the relationship between problematic Internet use, time spent online and personality traits, and none among a French sample. These results suggest the importance of assessing the impact of personality traits on Internet use, particularly on time spent online, by differentiating results in terms of gender and online activities.

[Relationship between defense mechanism and therapeutic alliance]. / Relation entre mécanismes de défense et alliance thérapeutique.

INTRODUCTION: The therapeutic alliance can be defined as a collaborative relationship between the patient and the practitioner. It represents an essential component of the psychotherapeutic process (Ambresin et al., 2007; Cungi, 2006; Martin et al., 2000). Some authors suggest that a good alliance can have a favorable impact on the therapeutic success (Barber et al., 2000; Hubble, Duncan, & Miller 1999; Horvath & Luborsky, 1993; Horvath & Symonds, 1991). This alliance can be influenced by psychological and behavioral factors (Cungi, 2006) Thus, some defense mechanisms could prevent change or, on the contrary could facilitate adaptation (Ambresin et al., 2007) and have an impact on the therapeutic success (Muris & Merckelbach, 1996). However, the relationship between therapeutic alliance and defense mechanisms represents an insufficiently explored field (Ambresin et al., 2007; Cungi, 2006). The aim of the present study was to examine the relationship between therapeutic alliance and twenty defense mechanisms in a sample of French psychiatric patients, by differentiating results in men and women. We also examined the positive and the negative therapeutic alliance. METHOD: Sixty patients aged from 18 to 58 (M=41.50; SD=11.03) completed the French versions of the Defense Style Questionnaire-40 (DSQ-40) and the Helping Alliance questionnaire-II (HAq-II). RESULTS: Therapeutic alliance was significantly associated with each defense style: mature (0.62), neurotic (0.45) P<0.01and immature (0.27) p<0.05. The mature defense style was a significant predictor of therapeutic alliance (R(2) adj=36, F=12.39, ß=0.65, P<0.01) and of positive therapeutic alliance (R(2) adj=36, F=12.34, ß=0.62, P<0.001). Among women, positive therapeutic alliance was significantly associated with all mature defenses, three neurotic defenses (reaction formation, pseudo-altruism, idealization) and four immature defenses (splitting, denial, somatization, passive aggression). Among men, three mature defenses were associated (anticipation, humor, sublimation), four neurotic (reaction formation, pseudo-altruism, idealization and undoing) and two immature (somatization and denial). The negative therapeutic alliance, in our total sample, was associated with two immature defenses (denial and dissociation). Among men, displacement was the only defense associated with negative alliance, among women no defenses was significant. DISCUSSION: These results highlight the relationship between therapeutic alliance and some defense mechanisms, like some authors have suggested (Ambresin et al., 2007; Bond & Perry, 2004; Bond, 2004). Moreover, some defenses appeared to be more associated with a positive or a negative therapeutic alliance, and could depend on the patient gender. CONCLUSION: The present study confirms the importance of taking into account the gender in the study of defense mechanisms, and to increase our knowledge about the relationship between therapeutic alliance and defense mechanisms.

One-step detection and genotyping of human papillomavirus in cervical samples by reverse hybridization.

This study describes a nonisotopic polymerase chain reaction-reverse hybridization-based method (PCR-RH) for the one-step detection and genotyping of anogenital human papillomavirus (HPV) in a microwell format. HPV DNA was amplified and labeled by PCR using GP5+/GP6+ primers. Labeled amplicons were hybridized to 20 HPV type-specific capture probes anchored to the surface of plastic microwells and detected by an immunoenzymatic assay. Assay sensitivity was <50 pg labeled amplicon, and no cross-reactivity was observed, as determined by hybridizing serial dilutions of labeled PCR products to either matched or mismatched capture probes. The assay was tested on 66 clinical samples (23 specimens with normal histology, I fibropapilloma, 26 cervical intraepithelial neoplasia grade 1 [CIN1], 9 CIN2, and 7 CIN3) and compared with a method based on restriction fragment length polymorphism (RFLP) of PCR products. PCR-RH and PCR-RFLP performed equally well on clinical samples. The overall HPV detection rate was similar: 65.1% (43/66) for PCR-RH and 57.6% (38/66) for PCR-RFLP. HPV DNA was found in all CIN2 and CIN3 samples by both methods; however, PCR-RH detected more positives among normal biopsy samples and CINI cases. Overall, there was good agreement between the two genotyping methods, but RH yielded fewer cases with undetermined HPV genotype.

A growth-constrained environment drives tumor progression invivo.

We recently have shown that selective growth of transplanted normal hepatocytes can be achieved in a setting of cell cycle block of endogenous parenchymal cells. Thus, massive proliferation of donor-derived normal hepatocytes was observed in the liver of rats previously given retrorsine (RS), a naturally occurring alkaloid that blocks proliferation of resident liver cells. In the present study, the fate of nodular hepatocytes transplanted into RS-treated or normal syngeneic recipients was followed. The dipeptidyl peptidase type IV-deficient (DPPIV(-)) rat model for hepatocyte transplantation was used to distinguish donor-derived cells from recipient cells. Hepatocyte nodules were chemically induced in Fischer 344, DPPIV(+) rats; livers were then perfused and larger (>5 mm) nodules were separated from surrounding tissue. Cells isolated from either tissue were then injected into normal or RS-treated DPPIV(-) recipients. One month after transplantation, grossly visible nodules (2--3 mm) were seen in RS-treated recipients transplanted with nodular cells. They grew rapidly, occupying 80--90% of the host liver at 2 months, and progressed to hepatocellular carcinoma within 4 months. By contrast, no liver nodules developed within 6 months when nodular hepatocytes were injected into the liver of recipients not exposed to RS, although small clusters of donor-derived cells were present in these animals. Taken together, these results directly point to a fundamental role played by the host environment in modulating the growth and the progression rate of altered cells during carcinogenesis. In particular, they indicate that conditions associated with growth constraint of the host tissue can drive tumor progression in vivo.

Massive liver replacement by transplanted hepatocytes in the absence of exogenous growth stimuli in rats treated with retrorsine.

A strategy for hepatocyte transplantation was recently developed whereby massive replacement of the recipient liver is achieved after a combined treatment with retrorsine, a pyrrolizidine alkaloid, and partial hepatectomy. We now investigated whether liver repopulation could occur in this animal model in the absence of any exogenous growth stimuli (eg, partial hepatectomy) for the transplanted cells. Dipeptidyl-peptidase type IV-deficient (DPPIV-) rats were used as recipients. Rats were given two injections of retrorsine (30 mg/kg each, 2 weeks apart), followed by transplantation of 2 x 10(6) hepatocytes isolated from a normal, syngeneic, DPPIV+ donor. At 2 weeks after transplantation, clusters of DPPIV+ hepatocytes occupied 3.3 +/- 0.9% of host liver, increasing to 38.2 +/- 6.3% at 2 months, and to 65.9 +/- 8.8% at 5 months. By 1 year, >95% of the original hepatocytes were replaced by donor-derived cells. Serum parameters related both to hepatocyte function and integrity (including glucose, bilirubin, total proteins, cholinesterase, alanine aminotransferase, and alkaline phosphatase) were in the normal range in retrorsine-treated and repopulated animals. These results provide further insights toward developing strategies for effective liver repopulation by transplanted hepatocytes with reduced toxicity for the host and potential clinical applicability.

Early exposure to restraint stress enhances chemical carcinogenesis in rat liver.

This study examines the effect of a stress-associated condition on chemical hepatocarcinogenesis in the rat. Rats were given diethylnitrosamine (200 mg/kg. b.w., i.p.), followed, 1 week later, by three cycles of immobilization at room temperature. Two weeks after the last cycle they were treated according to the resistant hepatocyte protocol. At 4 weeks after selection, mean size of glutathione-S-transferase 7-7 positive foci/nodules was increased in the immobilized group (0.82+/-0.22 vs. 0.25+/-0.04 mm(2) in controls). Furthermore, at the end of 1 year 10/13 animals (77%) developed hepatocellular carcinoma in the former group, while only 6/14 (43%) incidence of cancer was found in controls. These results indicate that exposure to restraint stress early during carcinogenesis enhances the development of chemically-induced hepatocellular carcinoma in the rat.

[Detection and genetic typing of human papillomaviruses in cervical biopsies in Cagliari Province]. / Ricerca e tipizzazione genotipica del papillomavirus umano in biopsie cervicali nella provincia di Cagliari.

INTRODUCTION: Human Papillomavirus (HPV) infection is considered an important risk factor for the development of cervical carcinoma. The aim of this work was to detect and genotype HPV DNA in cervical lesions from our Province. METHODS: HPV DNA was amplified by polymerase chain reaction (PCR) and genotyped by restriction fragment length polymorphism (RFLP) analysis. A total of 101 biopsies (43 koilocytic atypias, 20 CIN1, 19 CIN2, 17 CIN3 and 2 squamous carcinomas) were analyzed. RESULTS: HPV DNA was found in 41.8% of koilocytic atypias, in 95.0% of CIN1 and 100% of CIN2 and higher grade lesions. Only high risk genotypes were found in CIN2-3 and invasive carcinomas. HPV 16 was the most prevalent type in both CIN1 and CIN2-3 and the only HPV type found in situ and invasive carcinomas. HPV type 51 was found in 21.0% of CIN1 but it was rare in CIN2 and absent in more advanced lesions.

[Simultaneous detection and typing of human papillomavirus in cervical biopsies using PCR-reverse hybridization]. / Simultanea ricerca e tipizzazione del papillomavirus umano in biopsie cervicali mediante PCR-ibridazione inversa.

INTRODUCTION: Genotyping of Human papillomavirus (HPV) is an important step in the clinical evaluation of the oncogenic risk associated with HPV infection of cervical mucosa. The purpose of this work was to develop a fast PCR-reverse-hybridization assay (PCR-RH) for the simultaneous detection and genotyping of anogenital HPVs. METHODS: HPV DNA from cervical biopsies was amplified by consensus primer-PCR. Digoxigenin-labeled PCR products were hybridized to type-specific probes anchored to the surface of plastic microwells and revealed by an ELISA system. RESULTS: The method was tested on 115 clinical samples (81 koilocytic atypias, 11 CIN1, 10 CIN2, 12 CIN3 and 1 squamous carcinoma). HPV DNA was found in 56.7% koilocytic atypias, in 90.9% of CIN1 and in 100% of CIN2 and higher-grade lesions. Thus, PCR-RH is sensitive, rapid, easy-to-perform and readily applicable to the routine analysis of a large number of samples.

Liver regeneration in response to partial hepatectomy in rats treated with retrorsine: a kinetic study.

BACKGROUND/AIM: We have designed an experimental model in which transplantation of normal hepatocytes into rats previously treated with retrorsine (a naturally-occurring pyrrolizidine alkaloid) results in near-complete replacement of the recipient liver by donor-derived cells. Two/thirds partial hepatectomy was found to be essential for this process to occur. To probe this finding, in the present study we describe the kinetics of liver regeneration in response to partial hepatectomy in rats given retrorsine. METHODS: Six-weeks-old male Fisher 344 rats received retrorsine (2 injections of 30 mg/kg each, i.p., 2 weeks apart), or the vehicle. Four weeks after the last injection, partial hepatectomy was performed and rats were killed at 1, 2, 3, 6, and 15 days thereafter. RESULTS: At time zero, i.e. prior to partial hepatectomy, liver weight and total liver DNA content were significantly lower in retrorsine-treated animals compared to controls (DNA content: 19.2+/-1.7 vs. 25.7+/-1.1 mg/liver). Diffuse megalocytosis (enlarged hepatocytes) was present in the group exposed to retrorsine. By day 3 post-partial hepatectomy liver DNA content in control animals had more than doubled compared to day 1 values (20.2+/-1.5 vs. 8.8+/-1.2), while very little increase was seen in retrorsine-treated rats at the same time points (7.6+/-0.4 vs. 6.1+/-0.2). At 2 weeks after partial hepatectomy, total DNA content returned close to normal levels in the control group (26.9+/-1.0 mg/liver); however, the value was still very low in animals receiving retrorsine (9.1+/-0.7). Data on BrdU labeling were consistent with this pattern and indicated that DNA synthesis following partial hepatectomy was largely inhibited in the retrorsine group. Similarly, no mitotic response was observed in hepatocytes following partial hepatectomy in animals exposed to retrorsine. CONCLUSIONS: These results clearly indicate that retrorsine exerts a strong and persistent cell cycle block on hepatocyte proliferation. Further, these results are in agreement with the hypothesis that selective proliferation of transplanted hepatocytes in retrorsine-treated animals is dependent, at least in part, on the persistent cell cycle block imposed by the alkaloid on endogenous parenchymal cells.

Effect of fasting/refeeding on the incidence of chemically induced hepatocellular carcinoma in the rat.

Caloric restriction has been associated with a delay in the development of both spontaneous and induced neoplasia. In contrast, cycles of fasting/refeeding were shown by us and others to enhance the incidence of early lesions during chemical carcinogenesis in rat liver. The present, long-term study was undertaken to establish whether such a diffential effect would also extend to the later phases of cancer development, until the overt appearance of neoplasia. Male Fischer 344 rats were initiated with a single dose of diethylnitrosamine (DENA, 200 mg/kg i.p.) and starting 1 week later they were either exposed to three cycles of fasting (3 days) followed by refeeding (11 days) or were fed continuously. Seven weeks after DENA administration the rats were exposed to the resistant hepatocyte model of the liver tumor promotion protocol. All animals were killed 1 year after initiation. Incidence of hepatocellular carcinoma was 2-fold higher in the fasted/refed group compared with the controls (72 versus 36%). In addition, cancers were also larger and of higher histological grade in the former group, with one animal showing metastases to the lungs, while no metastases developed in control animals. Fasting caused a decrease in total liver DNA (from 25.2 +/- 1.1 to 16.5 +/- 1.1 mg after 3 days) which was associated with a decrease in hepatocyte labeling index and mitotic activity and high levels of single cell death (apoptosis). In contrast, a sharp increase in hepatocyte proliferation was observed on day 2 of refeeding and this was more pronounced in glutathione S-transferase 7-7 positive foci compared with surrounding liver (10.2 +/- 2.3 versus 4.6 +/- 0.8%). Such a proliferative wave was associated with a sharp decline in the incidence of cell death. It is concluded that fasting/refeeding performed early after initiation accelerates the development of chemically induced hepatocellular carcinoma in the rat.

Direct hyperplasia does not enhance the kinetics of liver repopulation in a new model of hepatocyte transplantation in the rat.

BACKGROUND/AIMS: We have recently developed a new model of extensive liver repopulation by transplanted hepatocytes following exposure to pyrrolizidine alkaloids. In the present study, the effect of 2/3 partial hepatectomy (PH) and that of a potent direct liver mitogen, lead nitrate, were compared in their ability to modulate the kinetics of liver repopulation. METHODS: Fischer 344 rats deficient in enzymatic activity for dipeptidyl-peptidase IV (DPPIV-) were used as cell transplantation recipients. They were given 2 doses of the pyrrolizidine alkaloid retrorsine (30 mg/kg, i.p.), 2 weeks apart, followed 2 weeks later by transplantation of 2 x 10(6) hepatocytes (via the portal vein), freshly isolated from a normal congeneic DPPIV+ donor. PH was carried out or a single injection of lead nitrate (100 micromol/kg, i.v.) was administered 2 weeks post-transplantation. Liver samples obtained at different time points post-treatment were processed histochemically for DPPIV activity. RESULTS: The percent of liver sections occupied by DPPIV+ hepatocytes was <1% at the time of PH or lead nitrate administration. In animals which underwent PH, it increased to 33.4+/-5.7% at 2 weeks and to 55.6+/-8.5% at 1 month. However, in animals receiving lead nitrate, these percentages were only 3.3+/-1.3% at 2 weeks and 16.2+/-3.9% at 1 month. Repeated injections of lead nitrate had no additional effect. Further experiments indicated that an acute mitogenic response to lead nitrate was present in transplanted cells, while resident hepatocytes were inhibited by retrorsine. CONCLUSIONS: These results indicate that direct mitogenic signals (such as those induced by lead nitrate), and compensatory signals (such as those elicited by PH), are not equally effective on kinetics of liver repopulation in this system. The possible reasons for these differential effects are discussed.

Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine.

Genetically marked hepatocytes from dipeptidyl peptidase (DPP) IV+ Fischer 344 rats were transplanted into the liver of DPPIV- mutant Fischer 344 rats after a combined treatment with retrorsine, a pyrrolizidine alkaloid that blocks the hepatocyte cell cycle, and two-thirds partial hepatectomy. In female rats, clusters of proliferated DPPIV+ hepatocytes containing 20 to 50 cells/cluster, mostly derived from single transplanted cells, were evident at 2 weeks, increasing in size to hundreds of cells per cluster at 1 month and 1000 to several thousand cells per cluster at 2 months, representing 40 to 60% of total hepatocyte mass. This level of hepatocyte replacement remained constant for up to 1 year, the duration of experiments conducted. In male rats, liver replacement occurred more rapidly and was more extensive, with transplanted hepatocytes representing 10 to 15% of hepatocyte mass at 2 weeks, 40 to 50% at 1 month, 90 to 95% at 2 months, 98% at 4 months, and 99% at 9 months. Transplanted hepatocytes were integrated into the parenchymal plates, exhibited unique hepatic biochemical functions, and fully reconstituted a normal hepatic lobular structure. The extensive proliferation of transplanted cells in this setting of persistent inhibition of resident hepatocytes represents a new general model to study basic aspects of liver repopulation with potential applications in chronic liver disease and ex vivo gene therapy.

Transient inhibition by orotic acid does not abolish the in vivo response of rat hepatocytes to a direct mitogen, lead nitrate.

BACKGROUND: Orotic acid (OA) is able to inhibit hepatocyte proliferation in vivo induced by 2/3 partial hepatectomy. The present studies were aimed at establishing: (i) whether OA also inhibits hepatocyte proliferation induced by a direct mitogen and, if so (ii) whether the stimulus provided by the mitogen is still expressed following transient inhibition by OA. METHODS/RESULTS: In the first experiment male Wistar rats were injected with either lead nitrate (100 mumol/kg, i.v.) or saline and 20 h later some animals receiving the mitogen were also implanted with a 400-mg OA tablet (as OA-methyl ester. i.p.). Multiple injections of 3H-thymidine were given to each rat (50 microCi each, 6 h apart, i.p.) until 2 h before killing. All groups were killed 3 days after the initial treatment. Results indicated that OA almost completely inhibited hepatocyte DNA synthesis and labelling induced by lead nitrate (e.g. labelling index was 1.9 +/- 0.5% in the saline-treated group, 44.7 +/- 4.0% in the lead nitrate group and 1.4 +/- 0.3% in the group receiving lead nitrate + OA). Based on the above results, in a second experiment rats were given a similar dose of lead nitrate and a subset of animals was implanted 20 h later with a 400-mg OA tablet, as previously described. Multiple doses of 3H-thymidine were again given to each rat (20 microCi each, 6 h apart) until 2 h before killing. Animals from both groups were killed at 3, 6 or 8 days after lead nitrate. Results indicated that, while at day 3 lead nitrate-induced DNA synthesis was effectively inhibited by OA, at day 6 the proliferative response was resumed in the group receiving OA. Cumulative labelling index over 6 days was 30.3 +/- 1.4 in rats given the mitogen alone and 52.1 +/- 2.2 in the group exposed to lead nitrate + OA. CONCLUSIONS: These data indicate that: (i) OA is also able to inhibit hepatocyte proliferation induced by a direct mitogen such as lead nitrate; this, in turn, suggests that its inhibitory effect is not unique to the stimulus elicited by partial hepatectomy. (ii) The proliferative response triggered by the mitogen is not abolished by the transient (3-4 days) inhibitory phase imposed by OA. Possible mechanisms underlying these effects are considered in the discussion.

Multiple genetic alterations in hamster pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinomas induced in the Syrian golden hamster (SGH) by N-nitrosobis(2-oxopropyl)amine share many similarities with the human disease, including mutations of the K-ras oncogene. In vitro carcinogenesis studies with immortal SGH pancreatic duct cells indicate that neoplastic transformation in this system can occur without mutational inactivation of p53 suppressor gene. In this study we extend the genetic analysis of the in vivo SGH model to increase the number of cases analyzed for the status of K-ras and to determine further the spectrum of alterations involved; we have studied the status of the p53, DCC, and Rb-1 suppressor genes and the status of the mdm2 oncogene, which can involve p53 indirectly. The partial SGH-coding sequence of mdm2 and DCC was determined. K-ras mutation in the second position of codon 12 was present in 17 of 19 (90%) of tumors. Immunohistochemistry and single strand conformation polymorphism analysis showed no evidence of p53 mutation in 21 tumors. RNase protection assays showed overexpression of mdm2 in 5 of 19 (26%) tumors. Semiquantitative reverse transcription-PCR analysis showed a complete or partial loss of DCC expression in 10 of 19 (53%) neoplasms and of Rb-1 (42%) expression in 8 of 19 tumors when compared to matched controls. Deregulation of these genes appears to be significant in SGH pancreatic carcinogenesis as indicated by their frequencies. However, the fact that 6 tumors showed either only a K-ras mutation or the absence of alterations of the 5 genes analyzed indicates that additional as yet unstudied or unknown genes are also involved in SGH pancreatic duct carcinogenesis.

Transformation of hamster pancreatic duct cells by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in vitro.

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen in laboratory animals. In the present study, in vitro transformation of spontaneously immortal hamster pancreatic duct cells following exposure to 20 mM NNK for 1,3,5 and 7 days is described. NNK imparted a dose-dependent and time-dependent toxicity to pancreatic duct cells in vitro. After NNK treatment, duct cells were grown either in complete duct medium (CDM) or in the absence of bovine pituitary extract, epidermal growth factor and Nu-serum (incomplete duct medium, IDM). Addition of NNK to the culture for 1 and 3 days did not affect the growth of the cells, whereas exposure of the cells for 5 and 7 days was inhibitory. One and 3 day NNK-treated cells were able to grow in the absence of growth factors and serum immediately after the treatment without any inhibition of growth. Untreated cells grew as a monolayer consisting of tightly packed polygonal cells with single nuclei. NNK treated cells also grew as a monolayer with numerous mitotic figures and multi-nucleated large cells. The doubling time between the untreated (16 h) and NNK-treated cells (14 h) was not significantly different prior to injection into the nude mice. NNK treated cells grown in IDM displayed anchorage independency in soft-agar. The tumorigenicity of the untreated and NNK treated cells (5 x 10(6)) was determined in nude mice. One and 3 day NNK-treated cells grown in CDM produced well-differentiated, mucinous tumors with a lower frequency (2/4 sites) and longer duration, but produced tumors at a higher frequency (4/4 sites) and shorter duration when grown in IDM. Five and 7 day NNK-treated cells grown in CDM did not produce any tumors; however, they produced tumors when grown in CDM followed by IDM (5/8 and 6/8 sites) with a shorter duration in nude mice. Analysis of DNA for k-ras mutation at codons 12, 13 and 61 showed G-A transition at codon 12 of the k-ras oncogene in tumor cells of 1 and 3 day NNK treatment. No mutation was detected in tumor cells from 5 and 7 day treatment.