Society of Toxicology Develops Learning Framework for Undergraduate Toxicology Courses Following the Vision and Change Core Concepts Model.

The Society of Toxicology announces the development of a Learning Framework (https://www.toxicology.org/education/docs/SOT-Toxicology-Learning-Objectives.pdf) for undergraduate toxicology that will facilitate the development and sharing of evidence-based teaching materials for undergraduate toxicology educators throughout the world. This Learning Framework was modeled on the "Vision and Change Report" (www.visionandchange.org), an effort of the National Science Foundation and American Association for the Advancement of Science defining Core Concepts and Core Competencies to inform undergraduate biology course design. Vision and Change (V&C) has gained national acceptance, becoming a foundation for 14 upper-level courses designed by professional life science scientific societies. The undergraduate toxicology Learning Framework includes 5 Core Concepts aligned with V&C that encompass the discipline of toxicology: Evolution; Biological Information, Risk and Risk Management; Systems Toxicology; and Pathways and Transformations for Energy and Matter. Underpinning the Core Concepts are Level 2 Toxicology Concepts, which are broad disciplinary categories, Level 3 Learning Objectives, which address specific learning goals, and Level 4 Example Learning Objectives and Case Studies, which provide examples of how content might be taught. Syllabi from more than 20 undergraduate toxicology courses and several undergraduate toxicology textbooks were surveyed to determine toxicology-related Learning Objectives. From these, undergraduate educators can design courses tailored to their institutional needs by selecting a subset of Learning Objectives. Publication of a Learning Framework for toxicology will enable integration into other disciplines and facilitate the development and sharing of evidenced-based teaching materials for toxicology to educators in allied disciplines. Ultimately this will expand toxicology's impact to a broader audience.

Effect of chronic glutathione deficiency on the behavioral phenotype of Gclm-/- knockout mice.

Enhanced oxidative stress or deficient oxidative stress response in the brain is associated with neurodegenerative disorders and behavioral abnormalities. Previously we generated a knockout mouse line lacking the gene encoding glutamate-cysteine ligase modifier subunit (GCLM). Gclm(-/-) knockout (KO) mice are viable and fertile, yet exhibit only 9-35% of wild-type levels of reduced glutathione (GSH) in tissues, making them a useful model for chronic GSH depletion. Having the global absence of this gene, KO mice--from the time of conception and throughout postnatal life--experience chronic oxidative stress in all tissues, including brain. Between postnatal day (P) 60 and P100, we carried out behavioral phenotyping tests in adults, comparing male and female Gclm(-/-) with Gclm(-/-) wild-type (WT) littermates. Compared with WT, KO mice exhibited: subnormal anxiety in the elevated zero maze; normal overall exploratory open-field activity, but slightly more activity in the peripheral zones; normal acoustic startle and prepulse inhibition reactions; normal novel object recognition with increased time attending to the stimulus objects; slightly reduced latencies to reach a random marked platform in the Morris water maze; normal spatial learning and memory in multiple phases of the Morris water maze; and significantly greater hyperactivity in response to methamphetamine in the open field. These findings are generally in agreement with two prior studies on these mice and suggest that the brain is remarkably resilient to lowered GSH levels, implying significant reserve capacity to regulate reactive oxygen species-but with regional differences such that anxiety and stimulated locomotor control brain regions might be more vulnerable.

In utero and lactational exposure to PCBs in mice: adult offspring show altered learning and memory depending on Cyp1a2 and Ahr genotypes.

BACKGROUND: Both coplanar and noncoplanar polychlorinated biphenyls (PCBs) exhibit neurotoxic effects in animal studies, but individual congeners do not always produce the same effects as PCB mixtures. Humans genetically have > 60-fold differences in hepatic cytochrome P450 1A2 (CYP1A2)-uninduced basal levels and > 12-fold variability in aryl hydrocarbon receptor (AHR)affinity; because CYP1A2 is known to sequester coplanar PCBs and because AHR ligands include coplanar PCBs, both genotypes can affect PCB response. OBJECTIVES: We aimed to develop a mouse paradigm with extremes in Cyp1a2 and Ahr genotypes to explore genetic susceptibility to PCB-induced developmental neurotoxicity using an environmentally relevant mixture of PCBs. METHODS: We developed a mixture of eight PCBs to simulate human exposures based on their reported concentrations in human tissue, breast milk, and food supply. We previously characterized specific differences in PCB congener pharmacokinetics and toxicity, comparing high-affinity-AHR Cyp1a2 wild-type [Ahrb1_Cyp1a2(+/+)], poor-affinity-AHR Cyp1a2 wild-type [Ahrd_Cyp1a2(+/+)], and high-affinity-AHR Cyp1a2 knockout [Ahrb1_Cyp1a2(-/-)] mouse lines [Curran CP, Vorhees CV, Williams MT, Genter MB, Miller ML, Nebert DW. 2011. In utero and lactational exposure to a complex mixture of polychlorinated biphenyls: toxicity in pups dependent on the Cyp1a2 and Ahr genotypes. Toxicol Sci 119:189-208]. Dams received a mixture of three coplanar and five noncoplanar PCBs on gestational day 10.5 and postnatal day (PND) 5. In the present study we conducted behavioral phenotyping of exposed offspring at PND60, examining multiple measures of learning, memory, and other behaviors. RESULTS: We observed the most significant deficits in response to PCB treatment in Ahrb1_Cyp1a2(-/-) mice, including impaired novel object recognition and increased failure rate in the Morris water maze. However, all PCB-treated genotypes showed significant differences on at least one measure of learning or behavior. CONCLUSIONS: High levels of maternal hepatic CYP1A2 offer the most important protection against deficits in learning and memory in offspring exposed to a mixture of coplanar and noncoplanar PCBs. High-affinity AHR is the next most important factor in protection of offspring.

In utero and lactational exposure to a complex mixture of polychlorinated biphenyls: toxicity in pups dependent on the Cyp1a2 and Ahr genotypes.

Polychlorinated biphenyls (PCBs) are persistent toxic pollutants occurring as complex mixtures in the environment. Humans are known genetically to have > 60-fold differences in hepatic cytochrome P450 1A2 (CYP1A2) levels and > 12-fold differences in aryl hydrocarbon receptor (AHR) affinity, both of which could affect PCB pharmacokinetics. Thus, we compared Ahr(b1)_Cyp1a2(+/+) high-affinity AHR wild-type, Ahr(d)_Cyp1a2(+/+) poor affinity AHR wild-type, Ahr(b1)_Cyp1a2(-/-) knockout, and Ahr(d)_Cyp1a2(-/-) knockout mouse lines. We chose a mixture of three coplanar and five noncoplanar PCBs to reproduce that seen in human tissues, breast milk, and the food supply. The mixture was given by gavage to the mother on gestational day 10.5 (GD10.5) and postnatal day 5 (PND5); tissues were collected from pups and mothers at GD11.5, GD18.5, PND6, PND13, and PND28. Ahr(b1)_Cyp1a2(-/-) pups showed lower weight at birth and slower rate of growth postnatally. Absence of CYP1A2 resulted in significant splenic atrophy at PND13 and PND28. Presence of high-affinity AHR enhanced thymic atrophy and liver hypertrophy in the pups. Concentrations of each congener were analyzed at all time points: maximal noncoplanar congener levels in maternal tissues were observed from GD18 until PND6, whereas the highest levels in pups were found between PND6 and PND28. Coplanar PCB concentrations were generally higher in Ahr(d)-containing pup tissues; these findings are consistent with earlier studies demonstrating the crucial importance of AHR-mediated inducible CYP1 in the gastrointestinal tract as a means of detoxication of oral planar polycyclic aromatic hydrocarbons.

Incorporating genetics and genomics in risk assessment for inhaled manganese: from data to policy.

Manganese is an essential nutrient, and a healthy human with good liver and kidney function can easily excrete excess dietary manganese. Inhaled manganese is a greater concern, because it bypasses the body's normal homeostatic mechanisms and can accumulate in the brain. Prolonged exposure to high manganese concentrations (>1mg/m(3)) in air leads to a Parkinsonian syndrome known as "manganism." Of greatest concern are recent studies which indicate that neurological and neurobehavioral deficits can occur when workers are exposed to much lower levels (<0.2mg/m(3)) of inhaled manganese in welding fumes. Consequently, researchers at NIOSH are conducting a risk assessment for inhaled manganese. Novel components of this risk assessment include an attempt to quantify the range of inter-individual differences using data generated by the Human Genome Project and experimental work to identify genetically based biomarkers of exposure, disease and susceptibility. The difficulties involved in moving from epidemiological and in vivo data to health-based quantitative risk assessment and ultimately enforceable government standards are discussed.

7H-dibenzo[c,g]carbazole metabolism by the mouse and human CYP1 family of enzymes.

Found in tobacco smoke, fossil fuel and other organic combustion products, 7H-dibenzo[c,g]carbazole (DBC) is a potent mouse lung carcinogen and potential human carcinogen. Although the first hydroxylation is critical for determining activation versus detoxication, the enzymes responsible for site-specific hydroxylation of DBC are not known. We found that DBC-DNA adduct levels are significantly higher in aromatic hydrocarbon receptor null Ahr(-/-) mice, suggesting that the induction of Aromatic hydrocarbon receptor (AHR)-regulated genes, such as those in the CYP1 family, decrease DBC genotoxicity. Using knockout mice for Cyp1a1, Cyp1a2 and Cyp1b1, we showed that the major CYP1 enzymes that metabolize DBC are CYP1A1 in beta-naphthoflavone (BNF)-induced liver, CYP1A2 in non-induced liver, CYP1B1 and CYP1A1 in induced lung and none in non-induced lung. DBC metabolism by the human CYP1 enzymes was examined in vitro using Supersomestrade mark. Each mouse CYP1, as well as each human CYP1, has a unique DBC metabolite profile. Comparison of the metabolite profile in BNF-induced mice suggested that CYP1A1 primarily generates 1-OH, 2-OH and (5 + 6)-OH-DBC, whereas CYP1A2 generates primarily (5 + 6)-OH-DBC and CYP1B1 primarily generates 4-OH-DBC. This was similar to that observed in the human CYP1 enzymes. Most importantly, lung CYP1B1 is associated with forming 4-OH-DBC, the most potent metabolite leading to DBC-DNA adducts. These studies suggest that for non-pulmonary routes of exposure (i.e. skin, gastric, i.p.), low hepatic expression of CYP1A2 and CYP1A1, together with high expression levels of lung CYP1B1 and CYP1A1, may define a phenotype for high susceptibility to carcinogens such as DBC.

Genetic differences in lethality of newborn mice treated in utero with coplanar versus non-coplanar hexabromobiphenyl.

Polybrominated biphenyl (PBB) exposure in humans is known to cause immunotoxicity and disorders related to the central nervous system. Coplanar PBBs bind to the aryl hydrocarbon receptor (AHR) in vertebrates. We compared the coplanar PBB, 3,3',4,4',5,5'-hexabromobiphenyl (cHBB), with its stereoisomer, the non-coplanar PBB, 2,2',4,4'6,6'-hexabromobiphenyl (ncHBB), using C57BL/6J (B6) inbred mice (having the high-affinity AHR) and congenic B6.D2-Ahr d mice (having the low-affinity AHR in a >99.8% C57BL/6J genetic background). Pregnant dams were treated i.p. with vehicle alone, cHBB, or ncHBB on gestational day 5 (GD 5). Unexpectedly, neonatal lethality within the first 72 h postpartum was significant in cHBB-treated B6 mice at doses as low as 2.5 mg/kg, whereas no deaths were seen in B6 pups whose mother had received ncHBB 100 mg/kg or in either B6.D2-Ahr d or Ahr(-/-) knockout mice whose mother had received cHBB 100 mg/kg. Histological and gross anatomical analyses of a battery of tissues in the mother or fetus at GD 18, as well as 24 h postpartum, revealed no significant differences, except for decreased thymus and spleen weights in cHBB-treated B6 GD 18 fetuses. Cross-fostering and genetics experiments confirmed the association of neonatal deaths principally with in utero (rather than lactational) exposure to cHBB, and also no paternal effect. For the end points of mouse neonatal lethality and immunotoxicity, cHBB appears to act through the high-affinity AHR receptor. Although dioxin in utero is well known to cause AHR-dependent cleft palate and hydronephrosis, cHBB did not; thus, chronic activation of the AHR appears to be necessary but not sufficient for AHR-mediated teratogenicity.

Oral benzo[a]pyrene in Cyp1 knockout mouse lines: CYP1A1 important in detoxication, CYP1B1 metabolism required for immune damage independent of total-body burden and clearance rate.

CYP1A1 and CYP1B1 metabolically activate many polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene, to reactive intermediates associated with toxicity, mutagenesis, and carcinogenesis. Paradoxically, however, Cyp1a1-/- knockout mice are more sensitive to oral benzo[a]pyrene exposure, compared with wild-type Cyp1a1+/+ mice (Mol Pharmacol 65:1225, 2004). To further investigate the mechanism for this enhanced sensitivity, Cyp1a1-/-, Cyp1a2-/-, and Cyp1b1-/- single-knockout, Cyp1a1/1b1-/- and Cyp1a2/1b1-/- double-knockout, and Cyp1+/+ wild-type mice were analyzed. After administration of oral benzo[a]pyrene (125 mg/kg/day) for 18 days, Cyp1a1-/- mice showed marked wasting, immunosuppression, and bone marrow hypocellularity, whereas the other five genotypes did not. After 5 days of feeding, steady-state blood levels of benzo[a]pyrene were approximately 25 and approximately 75 times higher in Cyp1a1-/- and Cyp1a1/1b1-/- mice, respectively, than in wild-type mice. Benzo[a]pyrene-DNA adduct levels were highest in liver, spleen, and marrow of Cyp1a1-/- and Cyp1a1/1b1-/- mice. Many lines of convergent data obtained with oral benzo[a]pyrene dosing suggest that: 1) inducible CYP1A1, probably in both intestine and liver, is most important in detoxication; 2) CYP1B1 in spleen and marrow is responsible for metabolic activation of benzo[a]pyrene, which results in immune damage in the absence of CYP1A1; 3) both thymus atrophy and hepatocyte hypertrophy are independent of CYP1B1 metabolism but rather may reflect long-term activation of the aryl hydrocarbon receptor; and 4) the magnitude of immune damage in Cyp1a1-/- and Cyp1a1/1b1-/- mice is independent of plasma benzo[a]pyrene and total-body burden and clearance. Thus, a balance between tissue-specific expression of the CYP1A1 and CYP1B1 enzymes governs sensitivity of benzo[a]pyrene toxicity and, possibly, carcinogenicity.

Theophylline pharmacokinetics: comparison of Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice, humanized hCYP1A1_1A2 knock-in mice lacking either the mouse Cyp1a1 or Cyp1a2 gene, and Cyp1(+/+) wild-type mice.

OBJECTIVES: Pharmacokinetics of theophylline was investigated in Cyp1(+/+) wild-type mice, Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice, and humanized hCYP1A1_1A2 mice lacking either the mouse Cyp1a1 or Cyp1a2 gene. METHODS AND RESULTS: Animals received a single dose of theophylline (8 mg/kg i.p.), either alone or pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 microg/kg i.p.) 24 h prior to theophylline. We found that mouse or human CYP1A2 is the predominant enzyme for theophylline metabolism, the contribution of mouse or human CYP1A1 to theophylline metabolism is negligible, and another TCDD-inducible enzyme plays a minor role in 1-methyluric acid and 1,3-dimethyluric acid formation as well as enhanced theophylline clearance from the body. The half-life of elimination from plasma was more than four times longer in Cyp1a2(-/-) than Cyp1(+/+) mice and more than 10 times different after TCDD pretreatment. In humanized hCYP1A1_1A2 mice lacking the mouse Cyp1a2 gene, the half-life of elimination from plasma was two to three times longer than that in Cyp1(+/+) mice and four to five times different after TCDD pretreatment. CONCLUSION: Replacement of mouse Cyp1a2 with a functional human CYP1A2 gene restored the ability to metabolize theophylline, and the metabolism changed to a humanized profile (i.e. 3-methylxanthine formation, not seen in the wild-type mouse). TCDD-pretreated hCYP1A1_1A2 Cyp1a2(-/-) mice exhibited enhanced theophylline metabolism and clearance, due to induction of the human CYP1A2 enzyme. Comparing the hCYP1A1_1A2 Cyp1a2(-/-) and wild-type mice with published clinical studies, we found theophylline clearance to be about 5 times and 12 times, respectively, greater than that reported in humans.

Oral exposure to benzo[a]pyrene in the mouse: detoxication by inducible cytochrome P450 is more important than metabolic activation.

The cytochrome P450 (CYP1A1) enzyme metabolically activates many polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), to DNA- and protein-binding intermediates that are associated with toxicity, mutagenesis, and carcinogenesis. As a result, it is widely accepted that CYP1A1 potentiates the toxicity of this class of chemicals. In distinct contrast, we show here that CYP1A1 inducibility is essential in the detoxication of oral BaP. We compared Cyp1a1(-/-) knockout mice, having the genetic absence of the CYP1A1 enzyme, with Cyp1a1(+/+) wild-type mice. At an oral BaP dose of 125 mg/kg/day, Cyp1a1(-/-) mice died within 30 days whereas Cyp1a1(+/+) mice displayed no outward signs of toxicity. The rate of BaP clearance was 4-fold slower in Cyp1a1(-/-) than Cyp1a1(+/+) mice. The cause of death in Cyp1a1(-/-) mice receiving oral BaP seemed to be immunotoxicity, including toxic chemical depression of the bone marrow; some toxic effects in Cyp1a1(-/-) mice were noted at a BaP dose as low as 1.25 mg/kg/day. DNA post-labeling studies demonstrated dramatically higher BaP-DNA adduct levels in all Cyp1a1(-/-) tissues assayed, with the exception of the small intestine, which is probably a major site of BaP metabolism in Cyp1a1(+/+) mice. Different BaP-DNA adduct patterns were also observed between the two genotypes receiving oral BaP. Despite previous studies in vitro and in cell culture that have shown a participatory role for CYP1A1 in BaP toxicity, the present data indicate that, in the intact animal, inducible CYP1A1 is extremely important in detoxication and protection against oral BaP toxicity.