Characterizing Adversity of Lysosomal Accumulation in Nonclinical Toxicity Studies: Results from the 5th ESTP International Expert Workshop.

Lysosomes have a central role in cellular catabolism, trafficking, and processing of foreign particles. Accumulation of endogenous and exogenous materials in lysosomes represents a common finding in nonclinical toxicity studies. Histologically, these accumulations often lack distinctive features indicative of lysosomal or cellular dysfunction, making it difficult to consistently interpret and assign adverse dose levels. To help address this issue, the European Society of Toxicologic Pathology organized a workshop where representative types of lysosomal accumulation induced by pharmaceuticals and environmental chemicals were presented and discussed. The expert working group agreed that the diversity of lysosomal accumulations requires a case-by-case weight-of-evidence approach and outlined several factors to consider in the adversity assessment, including location and type of cell affected, lysosomal contents, severity of the accumulation, and related pathological effects as evidence of cellular or organ dysfunction. Lysosomal accumulations associated with cytotoxicity, inflammation, or fibrosis were generally considered to be adverse, while those found in isolation (without morphologic or functional consequences) were not. Workshop examples highlighted the importance of thoroughly characterizing the biological context of lysosomal effects, including mechanistic data and functional in vitro readouts if available. The information provided here should facilitate greater consistency and transparency in the interpretation of lysosomal effects.

Common DNA methylation alterations in multiple brain regions in autism.

Autism spectrum disorders (ASD) are increasingly common neurodevelopmental disorders defined clinically by a triad of features including impairment in social interaction, impairment in communication in social situations and restricted and repetitive patterns of behavior and interests, with considerable phenotypic heterogeneity among individuals. Although heritability estimates for ASD are high, conventional genetic-based efforts to identify genes involved in ASD have yielded only few reproducible candidate genes that account for only a small proportion of ASDs. There is mounting evidence to suggest environmental and epigenetic factors play a stronger role in the etiology of ASD than previously thought. To begin to understand the contribution of epigenetics to ASD, we have examined DNA methylation (DNAm) in a pilot study of postmortem brain tissue from 19 autism cases and 21 unrelated controls, among three brain regions including dorsolateral prefrontal cortex, temporal cortex and cerebellum. We measured over 485,000 CpG loci across a diverse set of functionally relevant genomic regions using the Infinium HumanMethylation450 BeadChip and identified four genome-wide significant differentially methylated regions (DMRs) using a bump hunting approach and a permutation-based multiple testing correction method. We replicated 3/4 DMRs identified in our genome-wide screen in a different set of samples and across different brain regions. The DMRs identified in this study represent suggestive evidence for commonly altered methylation sites in ASD and provide several promising new candidate genes.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Progress to Date and Future Plans.

The International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice proposal (INHAND) has been operational since 2005. A Global Editorial Steering Committee manages the overall objectives of the project, and the development of harmonized terminology for each organ system is the responsibility of the Organ Working Groups, drawing upon experts from North America, Europe, and Japan. Great progress has been made with 9 systems published to date--respiratory, hepatobiliary, urinary, central/peripheral nervous systems, male reproductive and mammary, zymbals, clitoral, and preputial glands in Toxicologic Pathology and the integument and soft tissue and female reproductive in the Journal of Toxicologic Pathology as supplements and on a Web site--www.goReni.org. INHAND nomenclature guides offer diagnostic criteria and guidelines for recording lesions observed in rodent toxicity and carcinogenicity studies. The guides provide representative photomicrographs of morphologic changes, information regarding pathogenesis, and key references. The purpose of this brief communication is to provide an update on the progress of INHAND.

Liver hypertrophy: a review of adaptive (adverse and non-adverse) changes--conclusions from the 3rd International ESTP Expert Workshop.

Preclinical toxicity studies have demonstrated that exposure of laboratory animals to liver enzyme inducers during preclinical safety assessment results in a signature of toxicological changes characterized by an increase in liver weight, hepatocellular hypertrophy, cell proliferation, and, frequently in long-term (life-time) studies, hepatocarcinogenesis. Recent advances over the last decade have revealed that for many xenobiotics, these changes may be induced through a common mechanism of action involving activation of the nuclear hormone receptors CAR, PXR, or PPARα. The generation of genetically engineered mice that express altered versions of these nuclear hormone receptors, together with other avenues of investigation, have now demonstrated that sensitivity to many of these effects is rodent-specific. These data are consistent with the available epidemiological and empirical human evidence and lend support to the scientific opinion that these changes have little relevance to man. The ESTP therefore convened an international panel of experts to debate the evidence in order to more clearly define for toxicologic pathologists what is considered adverse in the context of hepatocellular hypertrophy. The results of this workshop concluded that hepatomegaly as a consequence of hepatocellular hypertrophy without histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a non-adverse reaction. This conclusion should normally be reached by an integrative weight of evidence approach.

Social impairments in Rett syndrome: characteristics and relationship with clinical severity.

BACKGROUND: While behavioural abnormalities are fundamental features of Rett syndrome (RTT), few studies have examined the RTT behavioural phenotype. Most of these reports have focused on autistic features, linked to the early regressive phase of the disorder, and few studies have applied standardised behavioural measures. We used a battery of standardised measures of behaviour and functioning to test the following hypotheses: (1) autistic behaviour is prominent throughout childhood in RTT; (2) autistic features are more salient in individuals with milder presentation; (3) severity of autistic behaviour is associated with a wider range of behavioural problems; and (4) specific MECP2 mutations are linked to more severe autistic behaviour. METHODS: Eighty MECP2 mutation-positive girls with RTT (aged 1.6-14.9 years) were administered: (1) the Screen for Social Interaction (SSI), a measure of autistic behaviour suited for individuals with severe communication and motor impairment; (2) the Rett Syndrome Behaviour Questionnaire (RSBQ), covering a wide range of abnormal behaviours in RTT; (3) the Vineland Adaptive Behavior Scales (VABS); and (4) a modified version of the Rett Syndrome Severity Scale (RSSS). Regression analyses examined the predictive value of age and RSSS on autistic behaviour and other behavioural abnormalities. T-tests further characterised the behavioural phenotype of individual MECP2 mutations. RESULTS: While age had no significant effect on SSI or RSBQ total scores in RTT, VABS Socialization and Composite scores decreased over time. Clinical severity (i.e. RSSS) also increased with age. Surprisingly, SSI performance was not related to either RSSS or VABS Composite scores. Autistic behaviour was weakly linked with the RSBQ Hand behaviour factor scores, but not with the RSBQ Fear/Anxiety factor. Clinical (neurological) severity did not predict RSBQ scores, as evidenced by the analysis of individual MECP2 mutations (e.g. p.R106W, p.R270X and p.R294X). CONCLUSIONS: Our data suggest that in RTT, autistic behaviour persists after the period of regression. It also demonstrated that neurological and behavioural impairments, including autistic features, are relatively independent of one another. Consistent with previous reports of the RTT phenotype, individual MECP2 mutations demonstrate complex associations with autistic features. Evidence of persistent autistic behaviour throughout childhood, and of a link between hand function and social skills, has important implications not only for research on the RTT behavioural phenotype, but also for the clinical management of the disorder.

Autism spectrum disorder in Down syndrome: cluster analysis of Aberrant Behaviour Checklist data supports diagnosis.

BACKGROUND: The diagnostic validity of autism spectrum disorder (ASD) based on Diagnostic and Statistical Manual of Mental Disorders (DSM) has been challenged in Down syndrome (DS), because of the high prevalence of cognitive impairments in this population. Therefore, we attempted to validate DSM-based diagnoses via an unbiased categorisation of participants with a DSM-independent behavioural instrument. METHODS: Based on scores on the Aberrant Behaviour Checklist - Community, we performed sequential factor (four DS-relevant factors: Autism-Like Behaviour, Disruptive Behaviour, Hyperactivity, Self-Injury) and cluster analyses on a 293-participant paediatric DS clinic cohort. The four resulting clusters were compared with DSM-delineated groups: DS + ASD, DS + None (no DSM diagnosis), DS + DBD (disruptive behaviour disorder) and DS + SMD (stereotypic movement disorder), the latter two as comparison groups. RESULTS: Two clusters were identified with DS + ASD: Cluster 1 (35.1%) with higher disruptive behaviour and Cluster 4 (48.2%) with more severe autistic behaviour and higher percentage of late onset ASD. The majority of participants in DS + None (71.9%) and DS + DBD (87.5%) were classified into Cluster 2 and 3, respectively, while participants in DS + SMD were relatively evenly distributed throughout the four clusters. CONCLUSIONS: Our unbiased, DSM-independent analyses, using a rating scale specifically designed for individuals with severe intellectual disability, demonstrated that DSM-based criteria of ASD are applicable to DS individuals despite their cognitive impairments. Two DS + ASD clusters were identified and supported the existence of at least two subtypes of ASD in DS, which deserve further characterisation. Despite the prominence of stereotypic behaviour in DS, the SMD diagnosis was not identified by cluster analysis, suggesting that high-level stereotypy is distributed throughout DS. Further supporting DSM diagnoses, typically behaving DS participants were easily distinguished as a group from those with maladaptive behaviours.

Respiration and parturition affected by conditional overexpression of the Ca2+-activated K+ channel subunit, SK3.

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.

Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids.

Prostaglandins play important and diverse roles in the CNS. The first step in prostaglandin synthesis involves enzymatic oxidation of arachidonic acid, which is catalyzed by prostaglandin H(PGH) synthase, also referred to as cyclooxygenase. We have cloned an inducible form of this enzyme from rat brain that is nearly identical to a murine, mitogen-inducible cyclooxygenase identified from fibroblasts. Our studies indicate that this gene, here termed COX-2, is expressed throughout the forebrain in discrete populations of neurons and is enriched in the cortex and hippocampus. Neuronal expression is rapidly and transiently induced by seizures or NMDA-dependent synaptic activity. No expression is detected in glia or vascular endothelial cells. Basal expression of COX-2 appears to be regulated by natural synaptic activity in the developing and adult brain. Both basal and induced expression of COX-2 are inhibited by glucocorticoids, consistent with COX-2 regulation in peripheral tissues. Our studies indicate that COX-2 expression may be important in regulating prostaglandin signaling in brain. The marked inducibility in neurons by synaptic stimuli suggests a role in activity-dependent plasticity.

Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites.

Neuronal activity is an essential stimulus for induction of plasticity and normal development of the CNS. We have used differential cloning techniques to identify a novel immediate-early gene (IEG) cDNA that is rapidly induced in neurons by activity in models of adult and developmental plasticity. Both the mRNA and the encoded protein are enriched in neuronal dendrites. Analysis of the deduced amino acid sequence indicates a region of homology with alpha-spectrin, and the full-length protein, prepared by in vitro transcription/translation, coprecipitates with F-actin. Confocal microscopy of the native protein in hippocampal neurons demonstrates that the IEG-encoded protein is enriched in the subplasmalemmal cortex of the cell body and dendrites and thus colocalizes with the actin cytoskeletal matrix. Accordingly, we have termed the gene and encoded protein Arc (activity-regulated cytoskeleton-associated protein). Our observations suggest that Arc may play a role in activity-dependent plasticity of dendrites.

Iron deficiency causes duodenum mucosal hyperplasia in male Wistar rats.

Administration of an iron-deficient diet to Wistar rats resulted within 14 days in reduced serum iron concentrations, a microcytic hypochromic anemia, characteristic for impaired hemoglobin synthesis, and an increase of duodenal epithelial cell proliferation. After 5 weeks of iron deficiency, hypochromic microcytic anemia and a clear increase of duodenum weight but no pronounced effects on cell proliferation was observed. Increased duodenum weights corresponded to significant increases in mucosal area, indicating a diffuse, simple mucosal hyperplasia. The sequence of events following iron depletion thus appears to be: (1) reduced serum iron levels, (2) induction of hypochromic microcytic anemia, (3) increased duodenal epithelial cell proliferation, and (4) increased duodenal weight (increased mucosal area). Iron deficiency anemia was rapidly reversible after a 2-week recovery period. However, increased duodenum weights were still noted at that time. Intramuscular iron supplementation in animals fed with iron-deficient diet maintained body iron levels not below normal values, and neither anemia nor increased duodenum cell proliferation were detected after 14 days. A 5-week iron supplementation period resulted in slightly increased serum iron values, and slightly decreased duodenal epithelial cell proliferation. Thus, increased duodenum mucosal hyperplasia was shown to be secondary to depletion of body iron and anemia and reflects an attempt to increase iron absorption to counteract iron deficiency.

The inhalation toxicity of di- and triethanolamine upon repeated exposure.

Systemic and respiratory tract (RT) toxicity of triethanolamine (TEA) was assessed in a 28-day nose-only inhalation study in Wistar rats (10animals/sex, concentrations: 0, 20, 100, 500mg/m3; 5 days/week, 6h/day). In two nose-only 90-day inhalation studies, with similar exposure design, Wistar rats were exposed to 0, 15, 150, 400mg/m3 diethanolamine (DEA) (DEA Study 1:13animals/sex, general subchronic study) and to 0, 1.5, 3, 8mg/m3 (DEA Study 2:10animals/sex) to specifically investigate respiratory tract toxicity. Only DEA induced systemic toxicity at or above 150mg/m3 (body and organ weight changes, clinical- and histo-pathological changes indicative for mild blood, liver, kidney and testicular effects). Neurotoxicity was not observed for both substances. Exposure to both substances resulted in laryngeal epithelial changes starting from 3mg/m3 for DEA (reversible metaplasia at the base of the epiglottis, inflammation at higher concentrations extending into the trachea) or from 20mg/m3 for TEA (focal inflammation, starting in single male animals). TEA appears to be less potent with respect to systemic toxicity and RT irritancy than DEA. The 90-day no adverse effect concentration" (NOAEC) for changes due to TEA exposure in the respiratory tract was 4.7mg/m3 derived by extrapolation from the NOAEC of the 28day study.

Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.

Development of a video-based evaluation tool in Rett syndrome.

This paper describes the development of a video-based evaluation tool for use in Rett syndrome (RTT). Components include a parent-report checklist, and video filming and coding protocols that contain items on eating, drinking, communication, hand function and movements, personal care and mobility. Ninety-seven of the 169 families who initially agreed to participate returned a videotape within 8 months of the first request. Subjects whose videos were returned had a similar age profile to those who did not provide a video but were more likely to have classical than atypical RTT. Evidence of the content and social validity and inter-rater reliability on 11 videos is provided. Video may provide detailed, objective assessment of function and behaviour in RTT.

Cycloxygenase-2 activity promotes cognitive deficits but not increased amyloid burden in a model of Alzheimer's disease in a sex-dimorphic pattern.

Administration of non-steroidal anti-inflammatory agents reduces the risk of developing Alzheimer's disease in normal aging populations, an effect that may occur from inhibition of the cyclooxygenases, the rate-limiting enzymes in the formation of prostaglandins. In this study, we investigated whether increased activity of cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase, potentiates disease progression in a transgenic mouse model of Alzheimer's disease. To study the functional effects of COX-2 activity, male and female bigenic mice (amyloid precursor protein with Swedish mutation [APPswe]-presenilin-1 protein with deletion of exon 9 [PS1dE9] and trigenic COX-2/APPswe-PS1dE9) were behaviorally tested +/-administration of the selective COX-2 inhibitor celecoxib. Behavioral testing included a three-trial Y maze that measures spatial working and recognition memories and an open field task that tested levels of hyperactivity. Overexpression of COX-2 in APPswe-PS1dE9 mice resulted in specific deficits in spatial working memory in female but not male mice. These sex-specific deficits were abolished by pharmacological inhibition of COX-2 activity. Importantly, COX-2-associated deficits were dependent on co-expression of all three transgenes since COX-2 single transgenic and APPswe-PS1dE9 bigenic mice showed normal memory. Quantification of amyloid plaque load and total Abeta 40 and 42 peptides did not reveal significant differences in trigenic versus bigenic mice treated with either vehicle or celecoxib. Taken together, these data indicate an interaction between the effects of COX-2 and Abeta peptides on cognition that occurs in a sex-specific manner in the absence of significant changes in amyloid burden. These findings suggest that pathological activation of COX-2 may potentiate the toxicity of Abeta peptides, particularly in females, without significantly affecting Abeta accumulation.

p53-dependent signaling sustains DNA replication and enhances clonogenic survival in 254 nm ultraviolet-irradiated human fibroblasts.

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1/SDI1/CAP20) exists in normal human fibroblasts in a quaternary complex with a cyclin, a cyclin-dependent kinase, and proliferating cell nuclear antigen. A model was proposed in which, during p53-mediated suppression of cell proliferation following treatment with 254 nm UV radiation (UVC), the enhanced expression of p21 might inhibit DNA replication by virtue of its interactions with proliferating cell nuclear antigen. To test this model, we examined the mechanisms of inhibition of DNA replication in diploid human fibroblasts that express human papillomavirus type 16 E6, which inactivates p53. E6-expressing cells were defective in G1 checkpoint responses of induction of p21 and G1 arrest after ionizing radiation-induced damage to DNA. Accordingly, E6-expressing cells were resistant to inactivation of single-cell colony formation by ionizing radiation. E6 cells also displayed normal S-phase checkpoint responses of inhibition and recovery of replicon initiation following exposure to ionizing radiation and normal ability to bypass pyrimidine dimers during DNA replication soon after UVC irradiation (i.e., postreplication repair). However, DNA replication 6 h after UVC exposure was significantly inhibited in E6 cells in comparison to isogenic controls. This failure to maintain DNA replication in S-phase cells was associated with enhanced sensitivity to inactivation of single-cell colony formation by UVC. These results indicate that the p53-induced p21 pathway is not involved in the immediate S-phase responses to radiation-induced DNA damage of inhibition of replicon initiation and translesion bypass. However, our results demonstrate that p53 and, conceivably, p21 contribute to the ability of normal human fibroblasts to sustain DNA replication activity and form colonies following UVC irradiation.

Role of postreplication repair in transformation of human fibroblasts to anchorage independence.

Cellular capacity for postreplication repair (PRR) and sensitivity to transformation to anchorage independence (AI) were quantified in normal foreskin and xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzo(a)pyrene-diol-epoxide I (BPDE-I). PRR is defined here as a collection of pathways that facilitate the replication of DNA damaged by genotoxic agents. It is recognized biochemically as the process by which nascent DNA grows longer than the average distance between two lesions in the DNA template. PRR refers more directly to the elimination of gaps in the daughter-strand DNA by mechanisms which remain to be determined for human cells, but which may include translesion replication and recombination. PRR was measured in diploid human fibroblasts by analysis of the dose kinetics for inhibition of DNA strand growth in carcinogen-treated cells. Logarithmically growing foreskin fibroblasts (NHF1) displayed D0 values of 4.3 J/m2 and 0.14 microM for the inhibition of DNA synthesis in active replicons by UV and BPDE-I, respectively. XP variant cells (CRL1162) exhibited corresponding D0 values of 1.5 J/m2 and 0.16 microM. The increased sensitivity to inhibition of DNA replication by UV in these XP variant fibroblasts (2.9-fold greater than normal) was mirrored by an enhanced frequency of transformation to AI. XP variant fibroblasts (CRL1162) were 3.2 times more sensitive to transformation to AI by UV than were the normal foreskin fibroblasts. As predicted by the PRR studies, both cell types exhibited similar frequencies of AI colonies induced by BPDE-I. Apparent thresholds were observed for induction of AI by UV (normal fibroblasts, 2.7 J/m2; XP variant fibroblasts, 0.3 J/m2) and BPDE-I (both, 0.05 microM). Doses of UV and BPDE-I above these thresholds produced proportional increases in the inhibition of DNA replication in operating replicons and in the induced frequency of anchorage-independent colonies. At doses of UV and BPDE-I that produced the same degree of inhibition of DNA strand growth, BPDE-I induced a greater number of cells capable of anchorage-independent growth than did UV in both normal and XP variant fibroblasts.

Reversible inhibition of rat hepatocyte proliferation by hydrocortisone and its effect on cell cycle-dependent hepatocarcinogenesis by N-methyl-N-nitrosourea.

The susceptibility of hepatocytes to carcinogenesis in vivo may be influenced by the phase of the cell cycle at which carcinogen-induced damage is incurred. In order to better understand this relationship, hepatic cell proliferation in juvenile male Fischer 344 rats was charted following a two-thirds partial hepatectomy. For hepatocytes, two distinct waves of DNA synthesis occurred which were followed after 6 to 8 hr by waves of mitotic cell division. In contrast to this kinetic pattern, when hydrocortisone was given after the partial hepatectomy, the initial waves of DNA synthesis and mitosis by hepatocytes were each delayed by about 15 hr. In rats not given hydrocortisone, susceptibility to heptocarcinogenesis by N-methyl-N-nitrosourea was greatest at 20 hr after partial hepatectomy when the peak fraction of proliferating hepatocytes was in the S phase. By shifting the time of onset of DNA synthesis, the hydrocortisone treatments also shifted the time with greatest sensitivity to N-methyl-N-nitrosourea, with hepatocytes in late G1 or S again the most susceptible. Numerous tumors were also induced by N-methyl-N-nitrosourea in extrahepatic tissues, including intestine, Zymbal's gland, nervous system, kidneys, odontogenic tissues, and peritesticular mesothelium. The results illustrate the importance of cell proliferation in carcinogenesis and further point to the specific sensitivity of certain cell cycle phases.

Cell cycle-dependent initiation of hepatocarcinogenesis in rats by (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo(a)py rene.

Male F344 rats were treated once with (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo(a)pyre ne (BPDE) at various times after a two-thirds partial hepatectomy. Diet containing 0.05% phenobarbital was fed subsequently to promote the development of hepatocellular neoplasms from initiated cells. This combined treatment caused an apparently continuous emergence of hepatocellular neoplasms between 31 and 60 weeks after initiation. Yields of hepatocellular neoplasms (adenomas and carcinomas) seen at 45 weeks after initiation varied according to the time of treatment with BPDE. Rats treated at 15 h after partial hepatectomy, when maximal fractions of proliferating hepatocytes were entering the S phase, displayed the greatest incidence and yield of hepatocellular neoplasms. Rats treated when hepatocytes were early in the prereplicative G1 phase demonstrated a significantly lower incidence and yield of hepatocellular neoplasms. No neoplasms were seen in livers of rats treated with BPDE without prior partial hepatectomy; this indicates the insensitivity or resistance of nonproliferating G0 hepatocytes to initiation by this chemical. The variation in yields of neoplasms could not be attributed to variation in binding of carcinogen to DNA or to rates of DNA repair. The risk of initiation of hepatocytes appeared to be correlated (r = 0.95) with the fraction of hepatocytes that were entering the S phase at the time of treatment with BPDE. These results reveal a significant cell cycle dependency for initiation of hepatocarcinogenesis by BPDE and show that proliferating hepatocytes are at greatest risk when early in the S phase. Quantitation of islands of hepatocellular alteration suggested a similar pattern of cell cycle-dependent sensitivity, although the cycle-dependent variation in island frequencies (1.4-fold) was less than the variation in yields of neoplasms (5-fold). However, 1300-5000 islands were estimated to appear for every one neoplasm that appeared. When viewed within the context of multistep carcinogenesis, and assuming that islands of altered hepatocytes represent a step in this process, then these results suggest that islands may represent populations of cells at least one step removed from neoplasia.

Defective postreplication repair in xeroderma pigmentosum variant fibroblasts.

Postreplication repair (PRR) was quantified in normal human fibroblasts and in xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzo[a]pyrene diol epoxide-I (BPDE-I). PRR may be defined as the elimination of discontinuities in the daughter-strand DNA and the replicative bypass of lesions in the DNA template. Pathways of PRR reduce the number of DNA growing points that are blocked at template lesions and increase the rate of growth of nascent DNA on damaged templates. Rates of DNA synthesis and strand growth were measured in solvent- and carcinogen-treated cells by velocity sedimentation analyses of radiolabeled nascent DNA in alkaline sucrose gradients. Logarithmically growing normal fibroblasts displayed D0 values of 6.3 J/m2 and 0.37 microM for the inhibition of DNA synthesis in active replicons by UV and BPDE-I, respectively. Under identical conditions, the XP variant cells exhibited D0 values of 1.5-2.0 J/m2 and 0.27-0.31 microM. Pulse-chase experiments were performed in cells synchronized at the beginning of the S phase. Normal and XP variant cells displayed inhibition of DNA strand growth by UV, with D0 values of 21.6 and 7.0 J/m2, respectively. The D0 values for the inhibition of DNA strand growth by BPDE-I were 0.85 microM for the normal cells and 0.62-0.79 microM for the XP variant cells. The inhibitions of DNA replication by UV and BPDE-I were also analyzed in terms of DNA lesion frequencies. Based on the D0 values for inhibition of DNA replication, we concluded that the XP variant cells express maximally 25-33% of the total PRR activity observed in normal fibroblasts after UV treatment. Conceivably, this deficiency in PRR activity results in the XP variant's increased risk of cancers induced by sunlight, because XP variant cells and normal fibroblasts are equally proficient in excision repair. Both normal and XP variant fibroblasts, however, displayed similar PRR activities in response to BPDE-I treatment.

p21CIP1 is not required for the early G2 checkpoint response to ionizing radiation.

We have previously reported that the immediate G2 checkpoint delay of normal human fibroblasts in response to ionizing radiation is correlated with inhibition of p34CDC2/cyclin B kinase activity. Here, we observed increased amounts of the cyclin-dependent protein kinase inhibitor p21CIP1 associated with p34CDC2/cyclin B protein complexes from irradiated normal human fibroblasts. Since wild-type p53 function is not required for the early G2 checkpoint response to ionizing radiation, we investigated whether a p53-independent induction of p21CIP1 was required for the G2 checkpoint. Early passage human fibroblasts expressing the E6 oncoprotein of human papilloma virus-type 16 (NHF4 E6) were analyzed. It has been demonstrated earlier than inactivation of wild-type p53 function in these cells by E6 protein does not alter their intact early G2 checkpoint response to gamma-rays. p21CIP1 was found to be undetectable in p34CDC2/cyclin B protein complexes and in total extracts from the E6-expressing cells, with or without exposure to ionizing radiation. These data indicate that p21CIP1 is not required for the immediate G2 checkpoint response and is not induced by a p53-independent pathway in G2 phase following exposure to gamma-rays.