Applying toxicogenomics in mechanistic and predictive toxicology.
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Animals↗
Biomedical subjects
Michael L Cunningham
Publications and source records attributed to Michael L Cunningham.
Surgical site infections after pediatric intracranial surgery for craniofacial malformations: frequency and risk factors.
OBJECTIVE: Surgical site infections (SSIs) after pediatric intracranial surgery for craniofacial malformations are relatively common and potentially serious nosocomial infections. Despite this, few studies have been published on this topic. We performed a retrospective study to determine the incidence of and factors associated with SSIs after intracranial surgery for craniofacial malformations at a single multidisciplinary craniofacial center during a 6-year period. METHODS: Retrospective chart review was performed on 254 patients who underwent 281 intracranial procedures during a 6-year period. Patients with SSIs (cases) were compared with those without SSIs (controls). National Nosocomial Infection Surveillance System criteria were used to identify cases of SSI. RESULTS: SSIs occurred in 9 (3.2%) of 281 intracranial procedures that took place during the study period. Factors associated with an SSI included a complicated diagnosis comprising a diagnosis of syndromic craniosynostosis, frontonasal dysplasia, or oblique facial cleft (odds ratio [OR], 13.0; 95% confidence interval [CI], 2.6-64.4); duration of surgery longer than 426 minutes (OR, 12.1; 95% CI, 2.4-59.9); closure of skin under tension (OR, 12.5; 95% CI, 3.0-52.6); use of bovine pericardium (OR and 95% CI undefined); more than four surgeons present during surgery (OR, 6.3; 95% CI, 1.2-32); pediatric intensive care unit stay longer than 2 days (OR, 10.8; 95% CI, 2.2-53.3); and use of a ventilator after surgery (OR, 4.8; 95% CI, 1.2-18.6). CONCLUSION: In this study, the presence of a complicated diagnosis and a number of other factors were associated with an SSI after pediatric intracranial surgery for craniofacial malformations. We speculate that a complicated diagnosis may be a marker for other factors that contribute to the risk of an SSI. Future studies investigating SSIs after intracranial surgery should consider these factors.
Child↗
MDCT diagnosis of the child with posterior plagiocephaly.
OBJECTIVE: In this article, we review the normal anatomy and development of the posterior skull base and describe distinguishing imaging features of the two most common causes of posterior plagiocephaly: posterior deformational plagiocephaly and unilateral lambdoid synostosis. We also describe three unusual cases of posterior plagiocephaly, including asymmetric premature fusion of the anterior and posterior intraoccipital synchondroses, with diagnoses enabled by volume-reformatted MDCT. CONCLUSION: Three-dimensional reformatted MDCT enables accurate diagnosis of common and rare causes of posterior plagiocephaly in children.
Child, Preschool↗
A symbolic shaped-based retrieval of skull images.
In this work, we describe a novel symbolic representation of shapes for quantifying skull abnormalities in children with craniosynostosis. We show the efficacy of our work by demonstrating an application of this representation in shape-based retrieval of skull morphologies. This tool will enable correlation with potential pathogenesis and prognosis in order to enhance medical care.
Algorithms↗
Unique patterns of gene expression changes in liver after treatment of mice for 2 weeks with different known carcinogens and non-carcinogens.
Previously we demonstrated that the mouse liver tumor response to the non-genotoxic carcinogens oxazepam and Wyeth-14,643 involved more differences than similarities in changes in early gene expression. In this study we used quantitative real-time PCR and oligonucleotide microarray analysis to identify genes that were up- or down-regulated in mouse liver early after treatment with different known carcinogens, including oxazepam (125 and 2500 p.p.m.), o-nitrotoluene (1250 and 5000 p.p.m.) and methyleugenol (75 mg/kg/day), or the non-carcinogens p-nitrotoluene (5000 p.p.m.), eugenol (75 mg/kg/day) and acetaminophen (6000 p.p.m.). Starting at 6 weeks of age, mice were treated with the different compounds for 2 weeks in the diet, at which time the livers were collected. First, expression of 12 genes found previously to be altered in liver after 2 weeks treatment with oxazepam and/or Wyeth-14,643 was examined in livers from the various chemical treatment groups. These gene expression changes were confirmed for the livers from the oxazepam-treated mice in the present study, but were not good early markers for all the carcinogens in this study. In addition, expression of 20 842 genes was assessed by oligonucleotide microarray [n = 4 livers/group, 2 hybridizations/liver (with fluor reversals)] and the results were analyzed using the Rosetta Resolver System and GeneSpring software. The analyses revealed that several cancer-related genes, including Fhit, Wwox, Tsc-22 and Gadd45b, were induced or repressed in unique patterns for specific carcinogens and not altered by the non-carcinogens. The data indicate that even if the tumor response, including molecular alterations, is similar, such as for oxazepam and methyleugenol, early gene expression changes appear to be carcinogen specific and seem to involve apoptosis and cell cycle-related genes.
Acetaminophen↗
Familial acromelic frontonasal dysostosis: autosomal dominant inheritance with reduced penetrance.
Acromelic frontonasal dysostosis (AFND) represents a subgroup of patients with frontonasal malformation with limb abnormalities including preaxial polydactyly and tibial hypoplasia. Previous case reports have suggested autosomal recessive inheritance, given parental consanguinity. However, no affected siblings have been described. Longitudinal clinical history is limited as many do not survive the first years of life. The molecular basis of AFND is not known. Previous investigators have proposed that AFND may result from a perturbation in the Sonic Hedgehog pathway. We present clinical and radiographic findings in two unrelated boys, ages 8 and 9 years, with AFND, one of whom has a family history suggesting dominant inheritance. A focused study of genetic marker data and candidate gene mutation analysis in this family is presented.
Abnormalities, Multiple↗
Capillary sieving electrophoresis/micellar electrokinetic capillary chromatography for two-dimensional protein fingerprinting of single mammalian cells.
We have developed a two-dimensional capillary electrophoresis method for the study of protein expression in single mammalian cells. The first-dimension capillary contains an SDS-pullulan buffer system to perform capillary sieving electrophoresis, which separates proteins based on molecular weight. The second-dimension capillary contains an SDS buffer for micellar electrokinetic capillary chromatography. After a 6-min-long preliminary separation, fractions from the first capillary are successively transferred to a second capillary, where they undergo further separation by MECC. Over 100 transfers and second-dimension separations are performed over an approximately 3.5-h-long period. We demonstrate this technology by generating protein fingerprints from single native MC3T3-E1 osteoprogenitor cells and MC3T3-E1 cells transfected with the human transcription regulator TWIST. We also present single-cell protein fingerprints from MCF-7 breast cancer cells before and following treatment to induce apoptosis.
Animals↗
Cranial suture simulator for ultrasound diagnosis of craniosynostosis.
BACKGROUND: In evaluating the effectiveness of ultrasound as a screening tool for craniosynostosis it was discovered that sonologists and sonographers needed more experience scanning and visualizing cranial sutures on ultrasound. OBJECTIVE: To create an ultrasound simulator to train radiologists and technologists to locate and recognize patent and fused cranial sutures in children. MATERIALS AND METHODS: The hypoechoic appearance of patent sutures was simulated by cutting lines into life-sized plastic doll heads and filling them with a commercial hypoechogenic material. Fused hyperechoic sutures were simulated by not cutting into the hard plastic region of a suture. The simulator's teaching value was evaluated on three radiology residents and three fellows. Subjects performed pre-training scans on unknown simulators, received feedback and an opportunity to scan a training simulator, and then performed post-training scans on random unknown simulators. Accuracy was recorded as percentage of correctly demonstrated sutures. RESULTS: The suture simulator reproduces the sonographic appearance of patent and fused cranial sutures. Accuracy of acquisition, interpretation, and overall diagnosis increased from 64 to 91%, 79 to 91%, 61 to 97%, respectively, between pre and post training scans. CONCLUSION: An ultrasound simulator can reproduce the appearance of patent and fused cranial sutures in children and can be used to train radiologists and technologists in the performance of a screening protocol.
Cranial Sutures↗
Progressive postnatal craniosynostosis and increased intracranial pressure.
Since its first description by Virchow in 1851, craniosynostosis has been known as a potentially serious condition resulting in premature fusion of skull sutures. Traditionally, craniosynostosis has been regarded as an event that occurs early in fetal development, resulting in a skull shape at birth that is determined by the suture or sutures involved. In recent years, a different form of craniosynostosis has been observed. Patients initially come to the attention of physicians because they exhibit midface hypoplasia or occasionally hypertelorism. The affected individuals all have a normal skull shape and open sutures in infancy but develop multiple-suture craniosynostosis postnatally, ultimately requiring surgical correction. These cases are significant because, although the patients do not initially display the physical manifestations of craniosynostosis, they frequently develop increased intracranial pressure, which can have devastating consequences. Unless these patients are recognized and vigilant follow-up monitoring is instituted at an early age, permanent impairment can result. A retrospective chart review study was conducted, and patients with multiple-suture craniosynostosis who developed symptoms of increased intracranial pressure were selected. The patients were divided into two groups, namely, those with normal sutures and/or head shape at birth (progressive craniosynostosis) (n = 15) and those with abnormal head shapes at birth (classic syndromic craniosynostosis) (n = 12). Clinical and radiological findings typically used to monitor the development of increased intracranial pressure were reviewed for both groups and compared. In addition, mutational analyses were performed. All patients with progressive postnatal craniosynostosis demonstrated clinical, radiological, or ophthalmological evidence of increased intracranial pressure, requiring skull expansion. Those patients displayed papilledema, anterior fontanelle bulge, and thumbprinting more often than did the patients with classic craniosynostosis. Thirteen of 15 patients were given the clinical diagnosis of Crouzon syndrome, which raises the question of whether such patients represent a subset of patients with this syndrome. Mutational analyses for the patients with progressive craniosynostosis demonstrated that, of 13 patients tested, 11 had mutations in exon 7 or 9 of FGFR2, which is a common site of mutations in Crouzon syndrome. The traditional indications of increased intracranial pressure used to monitor patients with classic craniosynostosis can be used to monitor patients with progressive postnatal craniosynostosis, particularly anterior fontanelle bulge, papilledema, and thumbprinting. It is thought that regular monitoring of these characteristics may lead to earlier diagnosis and allow for surgical intervention before the development of undesirable outcomes. It is important for clinicians to be aware of this group of patients, because any delay in diagnosis and treatment can result in severe consequences for the patients.
Child↗
Gene expression profiling of rat livers reveals indicators of potential adverse effects.
This study tested the hypothesis that gene expression profiling can reveal indicators of subtle injury to the liver induced by a low dose of a substance that does not cause overt toxicity as defined by conventional criteria of toxicology (e.g., abnormal clinical chemistry and histopathology). For the purpose of this study we defined this low dose as subtoxic, i.e., a dose that elicits effects which are below the detection of conventional toxicological parameters. Acetaminophen (APAP) was selected as a model hepatotoxicant because (1) considerable information exists concerning the mechanism of APAP hepatotoxicity that can occur following high doses, (2) intoxication with APAP is the leading cause of emergency room visits involving acute liver failure within the United States, and (3) conventional clinical markers have poor predictive value. Rats treated with a single dose of 0, 50, 150, or 1500 mg/kg APAP were examined at 6, 24, or 48 h after exposure for conventional toxicological parameters and for gene expression alterations. Patterns of gene expression were found which indicated cellular energy loss as a consequence of APAP toxicity. Elements of these patterns were apparent even after exposure to subtoxic doses. With increasing dose, the magnitude of changes increased and additional members of the same biological pathways were differentially expressed. The energy loss suggested by gene expression changes was confirmed at the 1500 mg/kg dose exposure by measuring ATP levels. Only by ultrastructural examination could any indication of toxicity be identified after exposure to a subtoxic dose of APAP and that was occasional mitochondrial damage. In conclusion, this study provides evidence that supports the hypothesis that gene expression profiling may be a sensitive means of identifying indicators of potential adverse effects in the absence of the occurrence of overt toxicity.
Acetaminophen↗
Expression of base excision DNA repair genes is a sensitive biomarker for in vivo detection of chemical-induced chronic oxidative stress: identification of the molecular source of radicals responsible for DNA damage by peroxisome proliferators.
Oxidative stress to DNA is recognized as one of the mechanisms for the carcinogenic effects of some environmental agents. Numerous studies have been conducted in an attempt to document the fact that chemical carcinogens that are thought to induce production of oxidants also cause the formation of oxidative DNA lesions. Although many DNA adducts continue to be useful biomarkers of dose/effect, changes in gene expression have been proposed to be a practical novel tool for studying the role of chemically induced oxidative DNA damage. Here, we hypothesized that expression of base excision DNA repair genes is a sensitive biomarker for in vivo detection of chemically induced chronic oxidative stress. To test this hypothesis, mice were treated with a known rodent carcinogen and peroxisome proliferator, WY-14,643 (500 ppm, 1 month). A number of end points that are commonly used to assess oxidative DNA damage were considered. Our data demonstrate that no difference in 8-oxoguanine, the number of abasic sites, or single strand breaks can be detected in genomic DNA from livers of control or WY-treated animals. However, a concordant marked induction of genes specific for the long-patch base excision DNA repair, a predominant pathway that removes oxidized DNA lesions in vivo, was observed in livers of WY-treated mice. Kupffer cell NADPH oxidase, and peroxisomes in parenchymal cells have been proposed as the potential sources of peroxisome proliferator-induced oxidants. The analysis of expression of base excision DNA repair genes was used to assess whether this biomarker of oxidative stress can be used to determine the source of oxidants. The data suggest that DNA-damaging oxidants are generated by enzymes that are induced after activation of peroxisome proliferator activator receptor alpha, such as those involved in lipid metabolism in peroxisomes, and are not the result of activation of NADPH oxidase in Kupffer cells. We conclude that expression of base excision DNA repair genes is a sensitive in vivo biomarker for chemically induced oxidative stress to DNA that can be successfully used for the identification of the molecular source of radicals responsible for DNA damage in vivo.
Animals↗
Human consumption of methyleugenol and its elimination from serum.
Under a mandate from the U.S. Congress, the National Toxicology Program (NTP) of the U.S. Department of Health and Human Services conducts animal bioassays for carcinogenicity of potentially toxic chemicals to which the U.S. population might be exposed. Methyleugenol, a natural as well as synthesized substance, was nominated for study because it is structurally similar to safrole, a known animal carcinogen. Methyleugenol was found to be a very potent multisite carcinogen in male and female F344/N rats and B6C3F1 mice at all doses tested in 2-year NTP bioassays using gavage dosing. For this reason, human toxicokinetic studies were added to the traditional NTP protocol. A commercial brand of gingersnaps was found by chemists at the Centers for Disease Control and Prevention to contain a relatively high concentration of methyleugenol. After thorough scientific and clinical review, and approval by a National Institutes of Health institutional review board for the protection of human subjects, a study was conducted with nine healthy adult male and female human volunteers. The volunteers were given 12 gingersnaps for breakfast. Blood was drawn immediately before the meal and at 15, 30, 60, and 120 min afterward. The mean +/- SD fasting level of methyleugenol in serum was 16.2 +/- 4.0 pg/g wet weight. Peak blood levels were found at 15 min (mean +/- SD, 53.9 +/- 7.3 pg/g wet weight), followed by a rapid decline; the half-life of elimination was about 90 min. The peak levels were within the range of methyleugenol blood levels in the U.S. population, as measured concurrently in a subset of nonfasting participants in the Third National Health and Nutrition Examination Survey (NHANES III).
Administration, Oral↗
Differential diagnosis of the trapezoid-shaped head.
OBJECTIVE: To recognize several conditions that result in a trapezoid head shape and review and contrast their various physical findings. METHODS: A detailed review of all patients seen in the Craniofacial Clinic at the Children's Hospital and Regional Medical Center in Seattle, Washington, over a 10-year period from 1991 to 2001, with the diagnosis of craniosynostosis and plagiocephaly was performed. During this period, 690 patients had a surgical correction of craniosynostosis, and 1537 patients had posterior plagiocephaly. RESULTS AND CONCLUSIONS: The shape of the head when viewed from the vertex position in an axial plane can be a significant diagnostic aid when evaluating a patient with plagiocephaly. Positional molding causes the vast majority of plagiocephaly. This deformational change results in a parallelogram-shaped head. A much more rare cause of plagiocephaly is lambdoid synostosis. With premature fusion of one of the lambdoid sutures, the head takes on a very characteristic trapezoid shape when viewed from the vertex. Unilateral coronal synostosis that occurs on the same side as either posterior positional molding or unilateral lambdoid synostosis will also result in the trapezoid head shape. Furthermore, on the rare occasion when anterior and posterior deformational plagiocephaly occurs on the same side, the trapezoid head shape may be the consequence. The choice of appropriate treatment modality requires systematic evaluation of the patient with a trapezoid-shaped head to determine the etiology of the deformation.
Craniosynostoses↗
Increased risk for developmental delay in Saethre-Chotzen syndrome is associated with TWIST deletions: an improved strategy for TWIST mutation screening.
The majority of patients with Saethre-Chotzen syndrome have mutations in the TWIST gene, which codes for a basic helix-loop-helix transcription factor. Of the genetic alterations identified in TWIST, nonsense mutations, frameshifts secondary to small deletions or insertions, and large deletions implicate haploinsufficiency as the pathogenic mechanism. We identified three novel intragenic mutations and six deletions in our patients by using a new strategy to screen for TWIST mutations. We used polymerase chain reaction (PCR) amplification with subsequent sequencing to identify point mutations and small insertions or deletions in the coding region, and real-time PCR-based gene dosage analysis to identify large deletions encompassing the gene, with confirmation by microsatellite and fluorescence in situ hybridization (FISH) analyses. The size of the deletions can also be analyzed by using the gene dosage assay with "PCR walking" across the critical region. In 55 patients with features of Saethre-Chotzen syndrome, 11% were detected to have deletions by real-time gene dosage analysis. Two patients had a translocation or inversion at least 260 kb 3' of the gene, suggesting they had position-effect mutations. Of the 37 patients with classic features of Saethre-Chotzen syndrome, the overall detection rate for TWIST mutations was 68%. The risk for developmental delay in patients with deletions involving the TWIST gene is approximately 90% or eight times more common than in patients with intragenic mutations.
Acrocephalosyndactylia↗
Primary pulmonary dysgenesis in velocardiofacial syndrome: a second patient.
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Chromosome Deletion↗
Ultrasound screening of the lambdoid suture in the child with posterior plagiocephaly.
BACKGROUND: The child with posterior plagiocephaly may have positional molding or unilateral lambdoid synostosis. Molding responds to conservative treatment, lambdoid synostosis requires surgical reconstruction. CT is diagnostic, but uses ionizing radiation, may need sedation, and the incidence of lambdoid fusion is only 2-3%. OBJECTIVE: The purpose of this prospective study was to evaluate ultrasound as a screening test of lambdoid sutural patency using CT as the reference standard. MATERIALS AND METHODS: In total, 41 children having head CT examinations were enrolled over 6 months. Of those, 29 were referred for abnormal head shape and suspected synostosis, of whom two had lambdoid fusion; 12 were referred for indications not related to head shape and found to have a normal study. Ultrasound scanning and interpretation of the lambdoid sutures was performed blinded to the CT reference standard. The lambdoid suture was read as patent or fused if a hypoechoic gap could or could not be seen between the hyperechoic calvarial bones, respectively. RESULTS: The mean sensitivity and specificity of ultrasound in distinguishing a patent from fused lambdoid suture by three blinded pediatric radiologists was 100% and 89%, respectively. CONCLUSIONS: Sonography of the lambdoid sutures shows excellent preliminary promise as a screening test of lambdoid sutural patency.
Cranial Sutures↗
Workshop overview: use of genomic data in risk assessment.
The completion of the Human Genome Project has provided the foundation to analyze the expression of all genes transcribed in a specific cell, as well as a reference against which to assess genetic variability and its impact on susceptibility. Recent advances in genomic technologies have set the stage for better understanding and predicting individual adaptive and toxicological responses after toxicant exposure. Thus, it is now possible to simultaneously assess expression levels for thousands of different genes using DNA microarrays, as well as assess posttranscriptional and posttranslational events using high throughput proteomics. Similarly, the risk of toxicant exposure-induced disease used to be estimated across populations with widely varying responses. However, new high-throughput genomic technologies have the potential to greatly improve the accuracy of risk assessment, allowing identification of sensitive subpopulations at risk and ultimately leading to personalized risk profiles based on genetic composition. Recognizing the importance these technologies will have on the practice of risk assessment and the development of regulatory guidelines, the Society of Toxicology sponsored a workshop to evaluate the current status of genomic research; the ethical, legal, and social issues associated with these approaches; and, in this context, how data derived from these technologies would impact risk assessment. This article summarizes the evaluation by experts in genomic research and risk assessment in the first workshop to provide a forum for interaction between these scientific disciplines.
Animals↗
Glucocorticoid-like effects of antihepatocarcinogen Rotenone are mediated via enhanced serum corticosterone levels: Molecular Fitting and Receptor Activation Studies.
BACKGROUND: Recent studies suggest that rotenone alters cell signal transduction pathways in a manner similar to glucocorticoids. Histological and biochemical markers of glucocorticoid effects in vivo, evaluated in our laboratories, provide further evidence for similarities in the activity of glucocorticoids and rotenone. The purpose of this study was to investigate the mechanism by which rotenone produces glucocorticoid-like effects. METHODS: Male B6C3F1 mice were treated for 7 days with rotenone (600 ppm in diet), the glucocorticoid antagonist RU486 (2 mg/kg/day, ip), corticosterone (2 mg/kg/day, ip), or both rotenone and RU 486. Control mice received drug-free diet and the vehicle (corn oil, ip). Following preservation in 10% neutral buffered formalin, tissues were embedded in paraffin. Sections were stained with hematoxylin, eosin, and were examined by light microscopy. Tissue sections were processed for in situ enzymatic end labeling of 3'-hydroxy-DNA strand breaks, a measure of apoptosis. Corticosterone was quantified in sera, using a solid phase radioimmunoassay kit. Cells (cell line 1470.2 derived from C127 mouse mammary adenocarcinoma cells) were transiently transfected with 5 &mgr;g of pLTRLuc and 1 &mgr;g of beta-Galactosidase expression vectors using a BTX square-wave pulser at 155 V, 4 pulses (40 ms each). Cells were then treated with dexamethasone, rotenone, or a mixture of both for 6 hr, harvested and assayed for luciferase and beta-Galactosidase activity. Using Root Mean Square (RMS) fit analysis (Alchemy trade mark, Tripose, Inc., St Louis, MO), we assessed possible structural similarities between rotenone and corticosterone, dehydrocorticosterone, glucocorticoid antagonists ZK 98.299, and RU 486. RMS fit was calculated by selecting three atoms in each of the molecules, followed by calculating the distance between these atoms. An RMS value of zero between two molecules indicates identical molecular characteristics. A positive value suggests diminished similarity with a value of 1 or higher excluding any such similarities. RESULTS: Although the stimulatory effect exerted by rotenone on hepatocellular apoptosis was in the opposite direction of that produced by the glucocorticoid antagonist RU 486, data suggested that rotenone does not directly activate the glucocorticoid receptor. Molecular fitting of rotenone to glucocorticoid receptor agonists and antagonists as well as examination of the transcriptional activation of a glucocorticoid-responsive reporter gene (Mouse MammaryTumorVirus) in response to rotenone indicated that it is highly unlikely that rotenone interacts directly with the glucocorticoid receptor. However, feeding male B6C3F1 mice a diet containing rotenone (600 ppm for 7 days) resulted in a 3-fold increase in serum levels of corticosterone relative to control animals. Corticosterone is the major glucocorticoid in rodents. CONCLUSION: Rotenone does not interact directly with the glucocorticoid receptor. Elevation of serum corticosterone levels in response to rotenone may explain the glucocorticoid-like effects of this compound, and may play a role in its anti-hepatocarcinogenic effect.
Journal Article↗