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We investigated whether learning an artificial language at 17 months was predictive of children's natural language vocabulary and grammar skills at 54 months. Children at 17 months listened to an artificial language containing non-adjacent dependencies, and were then tested on their learning to segment and to generalise the structure of the language. At 54 months, children were then tested on a range of standardised natural language tasks that assessed receptive and expressive vocabulary and grammar. A structural equation model demonstrated that learning the artificial language generalisation at 17 months predicted language abilities - a composite of vocabulary and grammar skills - at 54 months, whereas artificial language segmentation at 17 months did not predict language abilities at this age. Artificial language learning tasks - especially those that probe grammar learning - provide a valuable tool for uncovering the mechanisms driving children's early language development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Identifying patients with high genetic risk for cancer has important clinical ramifications, but hereditary cancer risk is often not identified because of testing barriers at both the provider and patient level. It is unknown how to best implement appropriate genetic testing and follow-up care into an operating primary care clinic. Implementation studies to date have been conducted in high resourced facilities under optimal conditions, often not at the clinic level. This study aims to compare and evaluate two population-wide engagement strategies for identifying members of a primary care clinic's population with a family or personal history of cancer and offering high-risk individuals genetic testing for cancer susceptibility mutations. The two engagement strategies are: 1) point of care screening (POC), conducted when a patient is scheduled for an appointment and 2) direct patient engagement (DPE), where outreach provides the patient an opportunity to complete screening online on their own time. The study will identify changes, problems, and inefficiencies in clinical flow during and after the implementation of risk assessment and genomic testing for cancer risk across primary care clinics. It will also evaluate the effects of the two engagement strategies on patient, provider, and clinic leader outcomes, including perceptions of benefits, harms, and satisfaction with the engagement strategy and process of cancer risk assessment and genetic testing, across gender, racial/ethnic, socioeconomic, and genetic literacy divides. Finally, the study will evaluate the cost-effectiveness and budget impact of each engagement strategy., (Copyright © 2020. Published by Elsevier Inc.)

Background: Humans spend most of the time in the postprandial state, yet most knowledge about high-density lipoproteins (HDL) derives from the fasted state. HDL protein and lipid cargo mediate HDL's antiatherogenic effects, but whether these HDL constituents change in the postprandial state and are affected by dietary macronutrients remains unknown., Objectives: This study aimed to assess changes in HDL protein and lipid composition after the consumption of a high-carbohydrate or high saturated fat (HSF) meal., Methods: We isolated HDL from plasma collected during a randomized, cross-over study of metabolically healthy subjects. Subjects consumed isocaloric meals consisting predominantly of either carbohydrate or fat. At baseline and at 3 and 6 hours postprandial, we quantified HDL protein and lipid composition by liquid chromatography-mass spectrometry., Results: A total of 15 subjects were included (60% female, aged 34 ± 15 years, body mass index: 24.1 ± 2.7 kg/m 2 ). Consumption of the HSF meal led to HDL enrichment in total lipid (P = .006), triglyceride (P = .02), and phospholipid (P = .008) content and a corresponding depletion in protein content. After the HSF meal, 16 of the 25 measured phosphatidylcholine species significantly increased in abundance (P values range from .027 to <.001), along with several sphingolipids including ceramides (P < .004), lactosylceramide (P = .023), and sphingomyelin-14 (P = .013). Enrichment in apolipoprotein A-I (P = .001) was the only significant change in HDL protein composition after the HSF meal. The high-carbohydrate meal conferred only minimal changes in HDL composition., Conclusion: Meal macronutrient content acutely affects HDL composition in the postprandial state, with the HSF meal resulting in enrichment of HDL phospholipid content with possible consequences for HDL function., (Copyright © 2020 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Background: Neuromuscular block using subjective monitoring and neostigmine reversal is commonly associated with postoperative residual neuromuscular block. We tested whether a protocol for the management of neuromuscular block that specified appropriate dosing and optimal neostigmine reversal was associated with a reduction in postoperative residual neuromuscular block., Methods: Rocuronium administration was guided by surgical requirements and based on the ideal body weight, with dose reductions for female sex and age >55 yr. Neostigmine was administered in adjusted doses after a train-of-four count of four was confirmed at the thumb. The protocol ensured a minimum of 10 min between neostigmine administration and tracheal extubation. We measured the postoperative residual neuromuscular block in patients undergoing abdominal surgery before and after introduction of the protocol. Pre-specified primary and secondary endpoints were incidence of postoperative residual neuromuscular block and severe postoperative residual neuromuscular block at the time of tracheal extubation, defined as normalised train-of-four ratios <0.9 and <0.7, respectively., Results: The incidence of postoperative residual neuromuscular block at tracheal extubation was 14/40 (35%) for patients managed according to the protocol compared with 22/38 (58%) for patients in the control group, odds ratio of 0.39, and 95% confidence interval of 0.14-1.07; P=0.068. The incidence of severe postoperative residual neuromuscular block at tracheal extubation showed a highly significant difference, odds ratio=0.06, and confidence interval of 0.00-0.43; P=0.001., Conclusions: The incidence of severe postoperative residual neuromuscular block was significantly reduced after the protocol was introduced. Given the limitations inherent in this before-and-after study, further research is needed to confirm these results., Clinical Trial Registration: NCT02660398., (Copyright © 2018 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Background: To test inter-rater reliability of the online Microscale Audit of Pedestrian Streetscapes (MAPS) tool between raters with varying familiarities of Phoenix, Arizona., Methods: The online MAPS tool, based on the MAPS in-field audit tool and scoring system, was used for audits. Sixty route pairs, 141 segment pairs, and 92 crossing pairs in Phoenix were included. Each route, segment or crossing was audited by two independent raters: one rater in Phoenix and the other in San Diego, California, respectively. Item, subscale scores, and total scores reliability analyses were computed using Kappa or intra-class correlation coefficient (ICC)., Results: The route overall score had substantial reliability (ICC: 0.832). Of the route subscale and overall scores, sixteen out of twenty had moderate to substantial reliability (ICC: 0.616-0.906), and the four subscales had fair reliability (ICC: 0.409-0.563). Sixteen out of twenty scores in segment and crossing sections demonstrated fair to substantial reliability (ICC: 0.448-0.897), and the remaining four had slight reliability (ICC: 0.348-0.364)., Conclusions: Most of the online MAPS items, subscales, and overall scores demonstrated fair to substantial reliability between raters with varied familiarities of the Phoenix area. Results support use of online MAPS to measure microscale elements of the built environment by raters unfamiliar with a region., Competing Interests: Conflicts of Interest: None.

Background: The influence of common disturbances seen in preeclampsia, such as changes in strong ions and weak acids (particularly albumin) on acid-base status, has not been fully elucidated. The aims of this study were to provide a comprehensive acid-base analysis in severe preeclampsia and to identify potential new biological predictors of disease severity., Methods: Fifty women with severe preeclampsia, 25 healthy non-pregnant- and 46 healthy pregnant controls (26-40 weeks' gestation), were enrolled in this prospective case-control study. Acid-base analysis was performed by applying the physicochemical approach of Stewart and Gilfix., Results: Mean [sd] base excess was similar in preeclamptic- and healthy pregnant women (-3.3 [2.3], and -2.8 [1.5] mEq/L respectively). In preeclampsia, there were greater offsetting contributions to the base excess, in the form of hyperchloraemia (BE(Cl) -2 [2.3] vs -0.4 [2.3] mEq/L, P<0.001) and hypoalbuminaemia (BE(Alb) 3.6 [1] vs 2.1 [0.8] mEq/L, P<0.001). In preeclampsia, hypoalbuminaemic metabolic alkalosis was associated with a non-reassuring/abnormal fetal heart tracing (P<0.001). Quantitative analysis in healthy pregnancy revealed respiratory and hypoalbuminaemic alkalosis that was metabolically offset by acidosis, secondary to unmeasured anions and dilution., Conclusions: While the overall base excess in severe preeclampsia is similar to that in healthy pregnancy, preeclampsia is associated with a greater imbalance offsetting hypoalbuminaemic alkalosis and hyperchloraemic acidosis. Rather than the absolute value of base excess, the magnitude of these opposing contributors may be a better indicator of the severity of this disease. Hypoalbuminaemic alkalosis may also be a predictor of fetal compromise., Clinical Trial Registration: clinicaltrials.gov: NCT 02164370., (© The Author 2015. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The present study explored whether perceived neighborhood environmental attributes associated with physical activity differ by neighborhood income. Adults aged 20-65 years (n=2199; 48% female; mean age=45 years; 26% ethnic minority) were recruited from 32 neighborhoods from the Seattle, WA and Baltimore, MD regions that varied in objectively measured walkability and neighborhood income. Perceived built and social environment variables were assessed with the Neighborhood Environment Walkability Scale. There were neighborhood income disparities on 10 of 15 variables. Residents from high-income neighborhoods reported more favorable esthetics, pedestrian/biking facilities, safety from traffic, safety from crime, and access to recreation facilities than residents of low-income areas (all p's <0.001). Low-income neighborhoods may lack amenities and safety attributes that can facilitate high levels of physical activity for both transportation and recreation purposes., (Copyright © 2011. Published by Elsevier Ltd.)

The identification of proteins aberrantly expressed in malignant B-cells can potentially be used to develop new diagnostic, prognostic or therapeutic targets. Proteomic studies of B-cell malignancies have made significant progress, but further studies are needed to increase our coverage of the B-cell malignant proteome. To achieve this goal we stress the advantages of using sub-cellular fractionation, protein separation, quantitation and affinity purification techniques to identify hitherto unidentified signalling and regulatory proteins. For example, proteomic analysis of B-cell plasma membranes isolated from patients with mantle cell lymphoma (MCL) identified the voltage-gated proton channel (HVCN1,[1]). This protein has now been characterised as a key modulator of B-cell receptor (BCR) signalling and abrogation of HVCN1 function could have a role in the treatment of B-cell malignancies dependent on maintained BCR signalling [2]. Similarly, proteomic studies on cell lysates from prognostic subtypes of CLL, distinguished by the absence (UM-CLL) or presence (M-CLL) of somatic hypermutation of the immunoglobulin heavy chain locus identified nucleophosmin 1 (NMP1) as a potential prognostic marker [3,4]. Thus, targeted proteomic analysis on selected organelles or sub-cellular compartments can identify novel proteins with unexpected localisation or function in malignant B-cells that could be developed for clinical purposes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

CCAAT enhancer-binding protein (CEBP) transcription factors play pivotal roles in proliferation and differentiation, including suppression of myeloid leukemogenesis. Mutations of CEBPA are found in a subset of acute myeloid leukemia (AML) and in some cases of familial AML. Here, using cytogenetics, fluorescence in situ hybridization (FISH), and molecular cloning, we show that 5 CEBP gene family members are targeted by recurrent IGH chromosomal translocations in BCP-ALL. Ten patients with t(8;14)(q11;q32) involved CEBPD on chromosome 8, and 9 patients with t(14;19)(q32;q13) involved CEBPA, while a further patient involved CEBPG, located 71 kb telomeric of CEBPA in chromosome band 19q13; 4 patients with inv(14)(q11q32)/t(14;14)(q11;q32) involved CEBPE and 3 patients with t(14;20)(q32;q13) involved CEBPB. In 16 patients the translocation breakpoints were cloned using long-distance inverse-polymerase chain reaction (LDI-PCR). With the exception of CEBPD breakpoints, which were scattered within a 43-kb region centromeric of CEBPD, translocation breakpoints were clustered immediately 5' or 3' of the involved CEBP gene. Except in 1 patient with t(14;14)(q11;q32), the involved CEBP genes retained germ-line sequences. Quantitative reverse transcription (RT)-PCR showed overexpression of the translocated CEBP gene. Our findings implicate the CEBP gene family as novel oncogenes in BCP-ALL, and suggest opposing functions of CEBP dysregulation in myeloid and lymphoid leukemogenesis.

Peroxisome proliferators (PPs) constitute a large and chemically diverse family of non-genotoxic rodent hepatocarcinogens that activate the PP-activated receptor alpha (PPARalpha). In order to investigate the hypothesis that PPs elicit their carcinogenic effects through the suppression of apoptosis, we established an in vitro assay for apoptosis using both primary rat hepatocytes and the FaO rat hepatoma cell line. Apoptosis was induced by transforming growth factor beta1 (TGFbeta1), the physiological negative regulator of liver growth. In this system, PPs could suppress both spontaneous and TGFbeta1-induced apoptosis. In order to understand the mechanisms of this regulation of apoptosis, we conducted microarray analysis followed by pathway-specific gene clustering in TGFbeta1-treated cells. After treatment, 76 genes were up-regulated and 185 were down-regulated more than 1.5-fold. Cluster analysis of up-regulated genes revealed three clusters, A-C. Cluster A (4h) was associated with 12% apoptosis and consisted of genes mainly of the cytoskeleton and extracellular matrix such as troponin and the proteoglycan SDC4. In cluster B (8h; 25% apoptosis), there were many pro- and anti-apoptotic genes such as XIAP, BAK1 and BAD, whereas at 16h (40% apoptosis) the regulated genes were mainly those of the cellular stress pathways such as the genes implicated in the activation of the transcription factor NFkappab. Genes found down-regulated in response to TGFbeta1 were mainly those associated with oxidative stress and several genes implicated in glutathione production and maintenance. Thus, TGFbeta1 may induce apoptosis via a down regulation of oxidant defence leading to the generation of reactive oxygen species. The ability of PPs to impact on these apoptosis pathways remains to be determined. To approach this question, we have developed a technique using laser capture microdissection of livers treated with the PP, clofibric acid coupled with gene expression array analysis. Results show that some of the key steps of the LCM process had an impact on the gene profiles generated. However, this did not preclude accurate determination of a PP-specific molecular signature. Thus, the choice of appropriate controls will ensure that meaningful gene expression analyses can be performed on tissue microdissected from the foci generated in clofibric acid treated livers. These data will allow the identification of specific genes that are regulated by PPs leading to changes in apoptosis and ultimately to tumours.

Predictive validity of each word from the McGill Pain Questionnaire (MPQ) has not been investigated in relation to pain etiology. The purpose of this study was to explore differences in the words used to describe nociceptive and neuropathic pain. Patients with lung cancer (N = 123) selected words from the 78 MPQ pain quality descriptors and indicated the corresponding pain site for each word. Using only the MPQ pain location, and the cancer and treatment data abstracted from medical records, each pain site was classified as nociceptive or neuropathic (etiology). Pain etiology and quality descriptors were tested for proportional differences with sensitivity, specificity, and predictive value calculated for statistically significant descriptors. Of the 457 pain sites, 343 were classified as nociceptive (75%), 114 as neuropathic (25%). Lacerating, stinging, heavy, and suffocating were selected for a significantly larger proportion of nociceptive sites whereas throbbing, aching, numb, tender, punishing, pulling, tugging, pricking, penetrating, punishing, miserable, and nagging were selected for a larger proportion of neuropathic sites. Ten words correctly predicted 78% of the sites with 81% sensitivity to nociceptive pain and 59% sensitivity to neuropathic pain. Interestingly, several pain quality descriptors (burning, shooting, flashing, tingling, itching, and cold) previously associated with neuropathic pain did not distinguish between neuropathic and nociceptive pain. Infrequent selection of many MPQ words and lack of neurological exam data in the medical records are possible explanations for inconsistency with previous literature. Prospective studies are needed to validate pain quality descriptors for nociceptive and neuropathic types of lung cancer pain.

Transforming growth factor-beta1 (TGF-beta1), is involved in controlling liver size, by inducing apoptotic cell death in hepatocytes. However the mechanism by which TGF-beta(1) induces caspase activation and cell death is unknown. Apoptosis can be initiated either by receptor-mediated (e.g. Fas/CD95) or non-receptor chemically mediated (stress-induced) processes. With Fas/CD95 receptor mediated cell death, a multi-protein complex (DISC) is assembled at the plasma membrane, which activates the downstream caspases and cell death. In stress-mediated apoptosis, a cytosolic DISC equivalent, the apoptosome is formed that activates the effector caspases. We have characterised this complex in THP.1 cells, and shown that this is a cytochrome c dependent process that induces the formation of an approximately 700 kDa apoptosome caspase processing complex. This is formed by oligomerisation of apoptotic protease-activating factor 1 (Apaf-1), and recruitment and processing of caspase-9. We have now shown that TGF-beta1-induced apoptosis also occurs via the release of cytochrome c and the subsequent oligomerisation of Apaf-1 into an approximately 700 kDa apoptosome complex. Our studies show that, even though TGF-beta1 induction of apoptosis is a receptor-mediated event, it operates through the mitochondrial/Apaf-1 caspase activation pathway that appears to act as a common execution pathway for many diverse apoptotic stimuli.

Chronic use of morphine affects the immune system and predisposes an individual to opportunistic infections. Macrophages play an important role in conferring a first line of defense against invading pathogens. Understanding the mechanisms by which morphine affects the functioning of macrophages would have significant therapeutic benefit in treatment against infections such as HIV and AIDS related syndromes. Two of the major cytokines secreted by activated macrophages are Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). Our studies show that morphine differentially modulates lipopolysaccharide (LPS) induced expression of IL-6 and TNF-alpha. Nanomolar concentrations of morphine synergize with LPS and augment the secretion of both IL-6 and TNF-alpha. However, at micromolar concentrations morphine inhibits LPS induced synthesis of IL-6 and TNF-alpha. Expression of both these cytokine genes is dependent on the activation of a transcription factor, NF kappa B. Interestingly, morphine treatment also modulated the activation of NF kappa B by LPS. Pretreatment with a low dose of morphine (nanomolar) resulted in an increase in NF kappa B activation. In contrast pretreatment with a high dose of morphine (micromolar) led to a significant decrease in NF kappa B activation. Furthermore unlike the augmentation which was naloxone reversible, the inhibition of NF kappa B by morphine was not reversed by naloxone, suggesting the involvement of a nonclassical opioid receptor.

Chronic treatment of mice with morphine affects the proliferation, differentiation, and function of immune cells. In the present study, we investigated the mechanism by which morphine inhibits phytohemagglutinin (PHA)/interleukin-1 (IL-1)-induced thymocyte proliferation. When compared to control cultures, morphine-treated thymocytes showed decreased steady-state levels of bioactive IL-2 and IL-2 mRNA. The reduced IL-2 concentration and reduced transcript levels correlated well with a decreased rate of synthesis of IL-2 mRNA as determined by nuclear runoff assays. Subsequent studies showed that morphine treatment affected transcriptional control elements of the IL-2 promoter by inhibiting the synthesis of a specific trans-activating nuclear factor, c-Fos. c-Fos mRNA levels measured by semiquantitative RT-PCR were significantly decreased in thymocytes following treatment with morphine and activation with PHA and IL-1. Under identical conditions, c-Jun mRNA levels were not altered. Electrophoretic mobility shift studies with the AP-1 consensus oligonucleotide showed significantly decreased levels of AP-1-protein complex formation in nuclear extracts prepared from morphine-treated cells. These studies demonstrate for the first time that opioid alkaloids such as morphine can impair mitogen-lymphokine-activated thymocyte proliferation by interfering with transcriptional activation of the IL-2 gene.

The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

Chemicals used in the treatment of cancer include several that are potent mutagens in a range of in vitro and in vivo assays. For some, genetic effects have also been demonstrated in humans, detected as chromosomal aberrations in peripheral lymphocytes. Because (1) many of these agents are confirmed mutagens, (2) humans are exposed to them in relatively high doses, and (3) an increasing number of early cancer victims are surviving to reproductive age, it is important that information be available on the genetic and reproductive hazards associated with exposure to these agents. Chlorambucil and melphalan are structurally related chemicals that are included in our efforts to identify and assess such hazards among cancer chemotherapy agents. To date, both have been reported to induce specific locus mutations in germ cells of male mice (Russell et al., 1989; Russel et al., 1992b) and melphalan is one of very few chemicals shown to induce such mutations in spermatogonial stem cells. More recently, both chemicals were found to have strong reproductive effects in female mice (Bishop and Generoso, 1995, in preparation). In the present studies, these chemicals were tested for the induction of dominant lethal mutations and heritable translocations in male mice. Both chemicals were found to have reproductive effects attributable to cytotoxicity in specific male germ cell stages and to induce dominant lethal mutations and heritable translocations in postmeiotic germ cells, particularly in mid to early stage spermatids. Thus, relatively extensive data are now available for assessing the genetic and reproductive hazards that may result from therapeutic exposures to these chemicals.

Patient autonomy is a guiding principle in medical decision-making in America. This is challenging when patients become mentally incapacitated and cannot express their preferences. Advance care planning (ACP) addresses this challenge. ACP is a deliberative and communicative process that helps people formulate and communicate preferences for future medical care in the event of mental incapacity. Advance directives are mechanisms for communicating and/or documenting ACP, and are either instructional (e.g. statement of treatment preferences in living wills) or proxy types (e.g. appointment of another person to speak on the patient's behalf). ACP discussions between patients and health care providers and patient-orientated educational ACP materials often ignore insights from 2 related activities, health promotion and human information processing. More effective ACP should occur with greater attention to the concepts of stages of change and self-efficacy, the Health Belief Model, and the necessary requisites for cognitive integration.

Results of continuing studies indicate that the mouse zygote and two-cell embryo stages are a window of susceptibility in the experimental induction of congenital anomalies with certain mutagenic agents. The mechanisms by which the mutagens initiate the pathogenesis of these developmental defects are not known. However, in certain cases there is evidence that a nonconventional, perhaps epigenetic, mechanism is involved. Detailed characterization of the spectrum of anomalies induced and comparison of responses at the various stages exposed allowed classification of the mutagens generally into two groups. One group is characterized by being effective only in the early stages of zygote development and capable of producing a relatively high incidence of fetal death and hydrops. The other group affects all of the zygote stages studied as well as the two cell-embryo, but does not increase the incidence of fetal death and hydrops. Except for hydrops, chemicals in the two groups do not differ in terms of the types of anomalies present among malformed live fetuses, which bear a resemblance to a subset of common, sporadic human developmental anomalies that are of unknown etiology. This similarity raises the possibility that certain human developmental defects may have their origins in events that happen in the zygote and early pre-implantation stages.

In a previous report (Generoso et al., 1985) it was shown that the two hybrid stocks of mice, (C3H/R1 x 101/R1)F1 and (SEC/R1 x C57BL/6)F1, differed in their responses to induction of chromosomal aberrations following exposure of the stem-cell spermatogonia to 500 R x 4 (4-week intervals) acute X-rays. The levels of response in the two stocks were paralleled by the effects on the length of the sterile period, which presumably results from stem-cell killing and repopulation. The present study was conducted in order to determine whether the differences between the two stocks in these parameters hold true also for other conditions of radiation exposure. Thus, comparative experiments were conducted using the following acute exposure regimens: 500 R single dose, 500 R + 500 R (24-h interval), 100 R + 900 R (24-h interval), and 500 R x 4 (8-week intervals). The endpoints measured were chromosome rearrangements in diakinesis/metaphase-I meiocytes, embryonic lethality in conceptuses, length of sterile period and testis weight. Trend analysis indicated that higher frequencies of chromosome rearrangements and embryonic lethality were recovered from (C3H/R1 x 101/R1)F1 than from (SEC/R1 x C57BL/6)F1 males, that there were no significant differences between stocks in testis weight reductions, and that there was no consistency in the direction of the significant differences that occurred in the length of the sterile period. A definitive conclusion regarding the possible association between induction of chromosomal aberrations and induction of cell killing awaits direct histological analysis of the stem-cell population.

A procedure is described for the isolation of highly enriched fractions of binucleated hepatocytes from rat liver. Liver cells isolated by EGTA and collagenase perfusion were initially subjected to centrifugal elutriation and second to flow cytometry coupled with Hoechst 33342 staining. The elutriation step yielded hepatocyte fractions which contained almost entirely mononuclear diploid cells and fractions enriched in binucleate hepatocytes. The fractions with the highest proportion of binucleated hepatocytes contained between 50 and 56% of these cells. Subsequent flow cytometric cell sorting yielded fractions which contained greater than 80% binucleated cells. These cells were viable in culture as demonstrated by the immunohistochemical detection of bromodeoxyuridine incorporation.

For some chemicals, induction of presumed dominant lethal mutations has been observed only in female mice and not in males. In those cases, questions arise as to (1) whether the increased embryonic mortality is due to genetic effects of the chemicals in the oocyte or, (2) is caused indirectly through maternal toxicity, and, if genetic, (3) the basis for the sex difference. These questions were studied using the compounds adriamycin and platinol. Neither compound induces dominant lethals in male germ cells, but both increased early embryonic mortality when females were treated prior to mating (treatment of maturing oocytes). Reciprocal zygote transfer experiments ruled out, either entirely or for the large part, maternal toxicity as the cause, and cytogenetic analysis of first-cleavage metaphases revealed high incidences of chromosomal aberrations. The results of both of these experiments thus provide evidence that the early embryonic mortality resulted from genetic effects induced in oocytes. Most interestingly, each compound produced unexpected types of chromosomal aberrations. Adriamycin produced deletions, rings, and presumed chromosome-type rearrangements. Platinol, on the other hand, produced a few chromatid-type aberrations, but the bulk of aberrations were characterized by disorganization of the chromatin, minute fragments, and thread-like chromatin bridges between fragments and chromosomes or between two or more chromosomes. The latter type of cytogenetic damage was observed primarily in the centromeric region. It is hypothesized that the female-specific dominant lethal effects of the two compounds are associated with the diffused state of the maturing oocyte chromosomes.

N,N'-Methylenebisacrylamide (MBA), a dimer of the monomeric acrylamide, was studied for induction of clastogenic effects in germ cells of male mice. It was found to be effective in inducing dominant-lethal mutations and heritable translocations in maturing sperm. The semisterile translocation carriers and their normal counterparts were used to determine the health impact of transmitted chromosomal rearrangements through anatomical analysis of their immediate descendants in utero. As expected, semisterility resulted primarily from embryonic death during the periimplantation stages presumably caused by sperm segregants with unbalanced chromosome complement fertilizing some of the eggs. Among conceptuses that survived to mid- and late-gestation stages, there was an increased incidence of developmental anomalies including fetal death and phenotypic defects. These anomalies are assumed to be caused by certain types of unbalanced segregants that are compatible with survival beyond the periimplantation period. This class of unbalanced segregants represent in humans a major health problem to the mother and her conceptus.

The rate of Cd accumulation by adult rat liver parenchymal cells in serum free primary culture in the presence of 100 microM CdCl2 was 10 times greater than that by non-parenchymal Kupffer cells. Addition of the monothiol chelating agents, cysteine and penicillamine, decreased Cd uptake in both cell types, the effect becoming more pronounced as the monothiol concentration was increased from 0.1 to 1.0 mM. These monothiols thus appear to reduce the availability of Cd for transport across the cell membrane. In contrast 1-10 molar excesses of the dithiol agents 2,3-dimercaptopropanol (BAL) or dithiothreitol (DTT) stimulated to variable extents the rate of Cd accumulation 2-10-fold in parenchymal cells and by over 100-fold in Kupffer cells. Supplementation of the media with 3% serum had little effect on the Cd accumulation in the presence of dithiols. Intravenous injection of Cd (0.05 mg/kg DCdCl2) with up to a 10-fold molar excess of cysteine or penicillamine had little effect on the hepatocellular Cd distribution. However Cd uptake by non-parenchymal cells was increased markedly by the simultaneous administration of BAL or DTT in 2 or 10 molar excess. Evidence is provided that these results may be partially explained by the endocytosis, particularly in Kupffer cells, of colloidal complexes of Cd which are formed with the dithiols but not the monothiols. These observations demonstrate that the physicochemical form of Cd determines its hepatocellular distribution which may be an important factor in the manifestation of Cd toxicity after thiol treatment.

When previously mated female mice were exposed to inhaled ethylene oxide at the time of fertilization of their eggs or during early pronuclear stage of the zygote (before DNA synthesis), a high incidence of mortality among conceptuses and of congenital abnormalities among both the dead and the surviving fetuses was observed. The developmental stage at which death occurred ranged from near the time of implantation to day 17 of gestation when examination of the uterine contents was performed. In comparison, midgestation and late fetal deaths were absent or minimal when the females were exposed either before mating or when conceptuses were in later zygotic stages (pronuclear DNA synthesis) or had reached the early two-cell stage. The random types of congenital abnormality observed and the remarkable stage-dependent sensitivity suggest a genetic basis for the response. The effects differ, both from genetic damages induced in premating germ cells, which lead only to death near the time of implantation, and from teratogenic damage, which leads to malformations only when exposure of embryos occurs during the period of major organogenesis.

Corn oil, sesame oil, peanut oil, or olive oil, injected intraperitoneally to female mice prior to insemination, increased the number of deciduomata. In many plant oil-treated females the number of implantation sites was markedly higher than corpora lutea count. These effects were not observed among females that were treated similarly with mineral oil or among females that were given corn oil via oral administration. Evidence indicates that these effects did not arise from induced dominant-lethal mutations but from decidual responses resulting from traces of oil reaching the uterine lumen.

Although heritable translocations are an important endpoint for the assessment of genetic risk from radiation, there has been a serious information gap with regard to their induction in spermatogonial stem cells, the most important cell stage in males for risk considerations. This led to uncertainty in estimating the magnitude of risk per unit exposure. Further, the relationship between the frequency of reciprocal exchanges scored by cytological analysis of the exposed male's meiocytes and the frequency of those transmitted to first-generation offspring needed to be re-examined. In order to fill in these gaps, two radiation studies, i.e., dose response and dose fractionation, were conducted on spermatogonial stem cells in which heritable and cytologically detected translocations were scored. The present data are by far the most extensive, to date, for heritable translocation induction in spermatogonial stem cells. The linearity of the rising portion of the dose-effect curve and the additivity of effects observed in the fractionation study allow a direct estimation of the number of transmissible translocations expected per unit exposure. Thus, the expected increase in heritable translocations per rad of acute X-rays is 3.89 X 10(-5) per gamete. The data also show a lack of consistency between cytologically and genetically scored translocations.

The mouse egg is ovulated with its nucleus arrested at the metaphase-II stage of meiosis. Sperm entry triggers the completion of the second meiotic division. It has been speculated that damage to the meiotic spindle of normally ovulated eggs at around the time of sperm entry could result in chromosome malsegregation and the death of conceptuses with numerical chromosome anomalies. This hypothesis was tested using nocodazole, a microtubule inhibitor. Nocodazole was administered either to maturing preovulatory oocytes or to normally ovulated eggs at one of the following stages: (1) the time of sperm entry, (2) early pronuclear stage, (3) pronuclear DNA synthesis, (4) prior to first cleavage division, (5) early 2-cell stage, or (6) prior to the second cleavage division. Little or no effect was observed for treatment times other than the time of sperm entry, when the egg is being activated to complete the second meiotic division. Remarkably high frequencies of embryonic lethality, expressed at around the time of implantation, were induced at this stage. Cytogenetic analysis of first cleavage metaphases of zygotes treated at the time of sperm entry revealed a high incidence of varied numerical chromosome anomalies, with changes in ploidy being predominant.

In the mouse, all autosomal monosomies and trisomies are lethal by the time of birth (Searle, 1981). To test whether ethanol ingested by females shortly after mating induces nondisjunction, as reported by Kaufman (1983) on the basis of cytological evidence, we attempted to determine whether the incidence of intrauterine death was affected by this treatment. The incidence of late death (day 11 postconception or later) was found to be significantly increased when ethanol was administered 2 h following a 30-min mating period, but not when the interval was shorter. Measurements of early death were not sensitive enough (because of the high control frequency) to show an effect of ethanol treatment. Limited cytological data showed an induced incidence of trisomy in line with the excess frequency of late death, but the trisomy incidence by itself was not significantly different from control. The overall level of effect in the present experiment was lower than that reported by Kaufman.

Ethylene oxide was studied for induction of dominant-lethal mutations and heritable translocations in male mice. The chemical was prepared in water and injected intraperitoneally. The dominant-lethal study was conducted using a single injection of 150 mg/kg (maximum tolerated dose); in the heritable translocation study males were injected daily on weekdays for 5 weeks with 60 or 30 mg/kg dose per day. Results clearly showed that ethylene oxide is effective in inducing dominant-lethal mutations and that the 4 stocks of untreated females used do not differ or may differ only slightly in the ability of their eggs to repair ethylene oxide-induced lesions in male germ cells. Increases in the frequencies of heritable translocations were also observed at the 2-dose levels. These frequencies did not deviate significantly from those expected on the basis of dose-square kinetics.

This study was conducted to estimate the frequency of transmitted reciprocal translocations per rad of exposure to alpha particles from [239Pu]citrate. Data indicate that the rate of induction of heritable translocations is related linearly to the duration of spermatogonia stem cell exposure. The estimated increase in heritable translocations per rad of exposure of the stem cell to alpha particles is in the range of 1.45-2.91 X 10(-5)/gamete.

Unbalanced (duplication/deficiency) sperm from balanced reciprocal translocations induced in spermatogonial stem cells of mice generally lead to embryonic lethality around the time of implantation. In a recent study (Generoso et al., 1985), it was found that the incidence of X-ray-induced embryonic lethality differed markedly between two hybrid stocks of irradiated male mice. A parallel difference in the frequencies of reciprocal translocations was observed cytologically in the meiocytes of irradiated males. In the present report, which is an adjunct to the study by Generoso et al. (1985), it was determined whether or not similar differences between the two stocks exist for congenital defects resulting from genetic damage to stem-cell spermatogonia. The results indicate not only an association between the frequencies of induced reciprocal translocations and congenital abnormalities, but also a parallel greater frequency of induced malformations in the (C3H X 101)F1 stock versus the (SEC X C57BL)F1 stock of males.

In an earlier study (Generoso et al., 1987), it was observed that the mutagen, ethylene oxide (EtO), produced remarkable increases in the incidence of developmental abnormalities and death of fetuses when early zygotic stages were exposed. This is a major finding in experimental induction of embryopathy, implicating genetic damage to the zygotes as the likely cause. In the subsequent study reported here, 3 other mutagens--ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), and triethylene melamine (TEM), were studied for embryopathic effects following exposure of dictyate oocytes, prefertilization oviducal eggs and sperm, early pronuclear zygotes, zygotes undergoing pronuclear DNA synthesis, and two-cell embryos. All 4 mutagens produced developmental abnormalities among living fetuses following exposure of early pronuclear zygotes (the only stage studied for this endpoint in this report). With respect to stage specificity and gestational timing of death of conceptuses, EMS and EtO on one hand and ENU and TEM on the other, are very similar to one another. EMS, like EtO, produced a high incidence of midgestation and late fetal deaths only in prefertilization oviducal eggs and sperm and in early pronuclear eggs. In contrast, ENU and TEM produced high losses of conceptuses in all postmating stages studied but death occurred primarily prior to or around the time of implantation. Thus, the frequency of induction and the expression of embryopathy, which ranged from early embryonic preimplantation and late fetal deaths to subtle fetal anomalies, are dependent upon the stage exposed and the mutagen used.

Ionizing radiation induces balanced reciprocal translocations in spermatogonial stem cells of mice. From cells carrying these rearrangements, which can be scored cytologically in the diakinesis-metaphase I stage, balanced normal, balanced translocated and unbalanced (duplication/deficiency) sperm can be produced. The relationship between expected (calculated from cytological data) and observed frequencies of embryonic lethality (presumably as a result of unbalanced sperm fertilizing the egg) following exposure of spermatogonial stem cells to X-rays was studied in two hybrid stocks. A marked difference in the incidence of induced embryonic lethality was found between the two stocks. Similarly, a difference in the cytological frequencies of translocations was also found, although smaller than that observed for embryonic lethality. Thus, it appears that the difference between the two stocks in the frequencies of embryonic lethality may be attributable both to processes occurring prior to metaphase I and to a difference in the rate of transmission of unbalanced chromosome constitutions.

The renal uptake and degradation of cadmium-thionein (Cd-Mt) were examined in relation to nephrotoxic effects. Studies with Cd-Mt labelled with [3H]cystine showed that both Cd2+ and tritium uptake in the kidneys were complete 4 h after injection. During this period, renal copper content doubles due to the replacement of thionein-bound Cd2+ with Cu2+. This process probably occurs in the blood, prior to metallothionein uptake. Once reabsorbed, the protein is rapidly degraded in the lysosomes at a rate in step with uptake. Consequently, at 4 h virtually all of the Cd-Mt was degraded, resulting in a high concentration of non-thionein bound Cd2+. This Cd2+ (approx. 11-12 micrograms Cd2+/g, i.e. 70% of the total renal Cd2+ burden) produces the toxic effects. Between 2-4 h, new thionein synthesis is initiated and Cd2+ gradually becomes bound as the metallothionein. By 4 days, 80% of the renal Cd2+ is bound to endogenous thionein. These studies demonstrate that even small amounts of non-thionein bound Cd2+ are toxic to the kidney.

Earlier studies in this laboratory revealed that ethylene oxide (EtO) or ethyl methanesulfonate (EMS) induced high frequencies of midgestation and late fetal deaths, and of malformations among some of the surviving fetuses, when female mice were exposed at the time of fertilization of their eggs or during the early pronuclear stage of the zygote. Effects of the two mutagens are virtually identical. Thus, in investigating the mechanisms responsible for the dramatic effects in the early pronuclear zygotes, the two compounds were used interchangeably in the experiments. First, a reciprocal zygote-transfer study was conducted in order to determine whether the effect is directly on the zygotes or indirectly through maternal toxicity. And second, cytogenetic analyses of pronuclear metaphases, early cleavage embryos, and midgestation fetuses were carried out. The zygote transplantation experiment rules out maternal toxicity as a factor in the fetal maldevelopment. Together with the strict stage specificity observed in the earlier studies, this result points to a genetic cause for the abnormalities. However, the cytogenetic studies failed to show structural or numerical chromosome aberrations. Since intragenic base changes and deletions may also be ruled out, it appears that the lesions in question induced in zygotes by the two mutagens are different from conventional ones and, therefore, could be a novel one in experimental mammalian mutagenesis. Alternatively, the mechanism could involve a non-mutational 'imprinting' process that caused changes in gene expression.

Benzo[a]pyrene was tested for induction of dominant-lethal mutations in germ cells of male mice. Clear-cut dominant-lethal effects were induced in middle and early spermatozoa. In contrast to the dominant-lethal observed the study showed no detectable increase in heritable translocations for these stages over the spontaneous level. Thus, the results provide another example of a chemical mutagen that is effective in inducing dominant-lethal mutations but relatively ineffective in inducing heritable translocations in male postmeiotic germ cells.

An accurate estimate of the error of misclassifying male translocation heterozygotes as normals is essential for the proper evaluation of results of the heritable translocation test in mice. The size of this error may vary from one laboratory to another depending, primarily, on the method or variation of the method used in screening for translocation heterozygotes. This report shows a way to estimate for misclassification errors involved in two methods, sequential and direct cytological analysis, of screening for translocation heterozygotes. A positive correlation was found between the degree of partial sterility of a male and the frequency of cells with multivalent configurations among his diakinesis-metaphase I cells. We interpreted this to confirm that the length of translocated chromosome segments has some influence on the proportion of unbalanced gametes in the ejaculate, presumably reflecting the frequency with which adjacent-1 and adjacent-1 segregations and 3-1 misdivisions occur.

DBCP was studied for dominant-lethal effects in male and female mice and for total reproductive effects in females. In males it was administered either intraperitoneally or subcutaneously while in females it was given only by the former route. No DBCP-related response was observed in either males or females indicating its ineffectiveness in inducing chromosomal aberrations or cytotoxicity in mouse germ cells. These findings differ markedly from the observations made in rats by other investigators. Thus, the probable existence of a species difference in germ cell response to DBCP has been strengthened by the availability of the present results. It should be noted, however, that only two stocks of male mice have been studied so far for dominant-lethal and germ cell cytotoxicity effects.

Degradation of the cadmium (Cd)-induced protein, metallothionein (MT) was studied in rat liver and kidney. The half-times (t1/2s) for degradation of the protein were found to vary according to the concentration of Cd in the tissues. In animals containing high Cd2+ concentrations, the t1/2 of degradation was significantly longer than in low Cd2+ animals. There were no significant differences in degradation times between liver and kidney. Purification of the MT by anion exchange chromatography revealed two species which had, in the liver at least, different degradation times. Since the metal content of the MTs did not vary throughout the time course of the experiment, it is proposed that the rate of degradation and hence the turnover of the protein is influenced by the ratio of Cd2+ to other bound cations (e.g. Zn2+ and Cu2+). Thus, the Cd/Zn ratio in the liver and the Cd/Cu ratio in the kidney determine the respective rates of degradation and hence turnover of the protein.