George Cruzan

CYP2F2-generated metabolites, not styrene oxide, are a key event mediating the mode of action of styrene-induced mouse lung tumors.

Styrene induces lung tumors in mice but not in rats. Although metabolism of styrene to 7,8-styrene oxide (SO) by CYP2E1 has been suggested as a mediator of styrene toxicity, lung toxicity is not attenuated in CYP2E1 knockout mice. However, styrene and/or SO metabolism by mouse lung Clara cell-localized CYP2F2 to ring-oxidized cytotoxic metabolite(s) has been postulated as a key metabolic gateway responsible for both lung toxicity and possible tumorigenicity. To test this hypothesis, the lung toxicity of styrene and SO was evaluated in C57BL/6 (WT) and CYP2F2⁻/⁻ knockout mice treated with styrene (400 mg/kg/day, gavage, or 200 or 400 mg/kg/day, ip) or S- or R-SO (200 mg/kg/day, ip) for 5 days. Styrene treated WT mice displayed significant necrosis and exfoliation of Clara cells, and cumulative BrdU-labeling index of S-phase cells was markedly increased in terminal bronchioles of WT mice exposed to styrene or S- or RSO. In contrast, Clara and terminal bronchiole cell toxicity was not observed in CYP2F2⁻/⁻ mice exposed to either styrene or SO. This study clearly demonstrates that the mouse lung toxicity of both styrene and SO is critically dependent on metabolism by CYP2F2. Importantly, the human isoform of CYP2F, CYP2F1, is expressed at much lower levels and likely does not catalyze significant styrene metabolism, supporting the hypothesis that styrene-induced mouse lung tumors may not quantitatively, or possibly qualitatively, predict lung tumor potential in humans.

Systemic Toxicity From Subchronic Dermal Exposure, Chemical Characterization, and Dermal Penetration of Catalytically Cracked Clarified Slurry Oil

Clarified slurry oil (CSO), the heavy residual fraction from the fluidized catalytic cracker, was applied to the shaven backs of groups of 10 male and 10 female Sprague-Dawley rats 5 days/week for 13 weeks at doses of 8, 30, 125, or 500 mglkglday, and to another group for 2 weeks at doses of 2000 mg/kg/day. The rats were fitted with cardboard Elizabethan collars to minimize the ingestion of the test material, which was applied undiluted and remained uncovered on the skin. A similar group of rats served as controls; they were treated in the same manner except that no CSO was applied to their skin. There was a dose-related mortality and depression of body weight gain in the rats treated with CSO at doses of 30 mg/kg/day or greater; none of the rats dosed at 2000 mg/kg/day survived more than 2 weeks. The primary target organs ofCSO toxicity were the liver, thymus, and bone marrow. The effects on the liver included increased weight (250% at 500 mg/kg/day), cholangiolitis, diffuse liver cell degeneration and hypertrophy, necrosis, fibrosis, decreased serum glucose, increased levels of alkaline phosphatase, aspartate aminotransferase, alanine amino transferase, bilirubin, and triglycerides. The thymus was found to be small and upon microscopic examination to be atrophic or hypoplastic. Erythroid hypo plasia was found in the bone marrow of some of the rats dosed at 30 mg/ kg/day and increased in severity with increasing dose. The erythroid hypoplasia was accompanied by a dose-related anemia. Even in the rats dosed at 8 mg/kg/day, very slight abnormalities in the bile ducts were observed upon microscopic examination of the liver. Chromato-graphic separation and analyses demonstrated that CSO contains about 58% 3- to 5-ring polycyclic aromatic hydrocarbons (PAHS) and ap proximately 8-10% carbazole derivatives. In vitro and in vivo skin penetration studies demonstrated that the carbazole materials penetrate through the skin to a considerable extent (about 44 %) ; less penetration was observed with 2- or 3-ring (8-13%) or 5-ring PAHs (3%).

Studies of styrene, styrene oxide and 4-hydroxystyrene toxicity in CYP2F2 knockout and CYP2F1 humanized mice support lack of human relevance for mouse lung tumors

Styrene (S) is lung tumorigenic in mice but not in rats. S and its alkene-oxidized metabolite styrene oxide (SO) were not lung toxic in CYP2F2(-/-) [knockout] mice, indicating S-induced mouse lung tumors are mediated through mouse-specific CYP2F2-generated ring-oxidized metabolite(s) in lung bronchioles. The human relevance of the CYP2F MOA was assessed by insertion of a human CYP2F1, 2A13, 2B6 transgene into CYP2F2(-/-) mice; CYP2F1 expression and activity were confirmed in the transgenic (TG) mice. No evidence of cytotoxicity or increased cell proliferation (BrdU labeling) was seen in TG mice treated with either S or SO (200mg/kg/day ip for 5days). In contrast to S and SO, 4HS (105mg/kg/day ip for 5days) increased BrdU labeling 5-10-fold in WT mice, <3-fold increase in KO mice and 2-4-fold in TG mice. The limited response of 4HS in KO and TG mice may result from intrinsic toxicity or from further metabolism; regardless of the MOA, these findings indicate that the CYP2F-mediated tumorigenic MOA in WT mice is not operative for S, SO, or for 4HS putatively derived from metabolism of S by CYP2F1 in humans, and thus S-induced mouse lung tumors are unlikely to be relevant to human risk.

Assessing molecular initiating events (MIEs), key events (KEs) and modulating factors (MFs) for styrene responses in mouse lungs using whole genome gene expression profiling following 1-day and multi-week exposures

ABSTRACT Styrene increased lung tumors in mice at chronic inhalation exposures of 20 ppm and greater. MIEs, KEs and MFs were examined using gene expression in three strains of male mice (the parental C57BL/6 strain, a CYP2F2(−/−) knock out and a CYP2F2(−/−) transgenic containing human CYP2F1, 2A13 and 2B6). Exposures were for 1‐day and 1, 4 and 26 weeks. After 1‐day exposures at 1, 5, 10, 20, 40 and 120 ppm significant increases in differentially expressed genes (DEGs) occurred only in parental strain lungs where there was already an increase in DEGs at 5 ppm and then many thousands of DEGs by 120 ppm. Enrichment for 1‐day and 1‐week exposures included cell cycle, mitotic M‐M/G1 phases, DNA‐synthesis and metabolism of lipids and lipoproteins pathways. The numbers of DEGs decreased steadily over time with no DEGs meeting both statistical significance and fold‐change criteria at 26 weeks. At 4 and 26 weeks, some key transcription factors (TFs) ‐ Nr1d1, Nr1d2, Dbp, Tef, Hlf, Per3, Per2 and Bhlhe40 ‐ were upregulated (|FC| > 1.5), while others ‐ Npas, Arntl, Nfil3, Nr4a1, Nr4a2, and Nr4a3 ‐ were down‐regulated. At all times, consistent changes in gene expression only occurred in the parental strain. Our results support a MIE for styrene of direct mitogenicity from mouse‐specific CYP2F2‐mediated metabolites activating Nr4a signaling. Longer‐term MFs include down‐regulation of Nr4a genes and shifts in both circadian clock TFs and other TFs, linking circadian clock to cellular metabolism. We found no gene expression changes indicative of cytotoxicity or activation of p53‐mediated DNA‐damage pathways. HighlightsStyrene response consistent with direct mitogenicity of Cyp2F2 and Nr4a signalingLonger term exposure show changes in circadian pathways.Changes in circadian pathways associated with Nr4a receptor family down‐regulationConsistent changes were seen only in wild type mice.No evidence of activation of p53‐mediated DNA‐damage or cell stress pathways

A Composite Model for Multiple Assays of Skin Irritation

A model for skin irritation was developed for simultaneous evalua tion of the influence on irritation of abrasion, occlusion, and dura tion of treatment and for fulfillment of requirements for labeling considerations under DOT, CPSC-FHSA, OSHA, and EEC. This model greatly reduces the number of animals required to address submissions under multiple agencies compared to performing each test separately. In this model, which we have called a Composite Skin Irritation test, a test material is placed on three pairs of intact and abraded sites on each rabbit; one pair of sites is occluded for 4 hours, one for 24 hours, and the other left unoccluded for 24 hours. Results are presented from 88 composite tests with 80 petroleum- related materials. For the materials tested, abrasion of the skin had no effect on the irritation response. Occlusion of the test site gener ally did not result in dramatic increases in response, except for petroleum refinery streams with a boiling range below 500° F. Exposure for 4 hours rather than 24 hours generally resulted in less irritation; however, for individual compounds, -the irritation from the 4-hour exposure could not be predicted from the response to the 24-hour exposure. Of the 80 materials tested, 12 would be labeled as skin irritants under CPSC guidelines, three under OSHA, and 20 under EEC. Of the 20 that would be labeled under EEC criteria, only seven would be labeled under CPSC criteria. At least for petroleum-related materials, results from skin irritation studies performed under one set of conditions cannot be used to predict the degree of irritation that would be produced under a dif ferent set of exposure conditions.

Editor’s Highlight: Complete Attenuation of Mouse Lung Cell Proliferation and Tumorigenicity in CYP2F2 Knockout and CYP2F1 Humanized Mice Exposed to Inhaled Styrene for up to 2 Years Supports a Lack of Human Relevance

Abstract Styrene is a mouse-specific lung carcinogen, and short-term mode of action studies have demonstrated that cytotoxicity and/or cell proliferation, and genomic changes are dependent on CYP2F2 metabolism. The current study examined histopathology, cell proliferation, and genomic changes in CD-1, C57BL/6 (WT), CYP2F2(−/−) (KO), and CYP2F2(−/−) (CYP2F1, 2B6, 2A13-transgene) (TG; humanized) mice following exposure for up to 104 weeks to 0- or 120-ppm styrene vapor. Five mice per treatment group were sacrificed at 1, 26, 52, and 78 weeks. Additional 50 mice per treatment group were followed until death or 104 weeks of exposure. Cytotoxicity was present in the terminal bronchioles of some CD-1 and WT mice exposed to styrene, but not in KO or TG mice. Hyperplasia in the terminal bronchioles was present in CD-1 and WT mice exposed to styrene, but not in KO or TG mice. Increased cell proliferation, measured by KI-67 staining, occurred in CD-1 and WT mice exposed to styrene for 1 week, but not after 26, 52, or 78 weeks, nor in KO or TG mice. Styrene increased the incidence of bronchioloalveolar adenomas and carcinomas in CD-1 mice. No increase in lung tumors was found in WT despite clear evidence of lung toxicity, or, KO or TG mice. The absence of preneoplastic lesions and tumorigenicity in KO and TG mice indicates that mouse-specific CYP2F2 metabolism is responsible for both the short-term and chronic toxicity and tumorigenicity of styrene, and activation of styrene by CYP2F2 is a rodent MOA that is neither quantitatively or qualitatively relevant to humans.

Strain-related differences in mouse lung gene expression over a two-year period of inhalation exposure to styrene: Relevance to human risk assessment

ABSTRACT Both CD‐1 and C57BL/6 wildtype (C57BL/6‐WT) mice show equivalent short‐term lung toxicity from exposures to styrene, while long‐term tumor responses are greater in CD‐1 mice. We analyzed lung gene expression from styrene exposures lasting from 1‐day to 2‐years in male mice from these two strains, including a Cyp2f2(−/−) knockout (C57BL/6‐KO) and a Cyp2F1/2A13/2B6 transgenic mouse (C57BL/6‐TG). With short term exposures (1‐day to 1‐week), CD‐1 and C57BL/6‐WT mice had thousands of differentially expressed genes (DEGs), consistent with changes in pathways for cell proliferation, cellular lipid metabolism, DNA‐replication and inflammation. C57BL/6‐WT mice responded within a single day; CD‐1 mice required several days of exposure. The numbers of exposure related DEGs were greatly reduced at longer times (4‐weeks to 2‐years) with enrichment only for biological oxidations in C57BL/6‐WT and metabolism of lipids and lipoproteins in CD‐1. Gene expression results indicate a non‐genotoxic, mouse specific mode of action for short‐term styrene responses related to activation of nuclear receptor signaling and cell proliferation. Greater tumor susceptibility in CD‐1 mice correlated with the presence of the Pas1 loci, differential Cytochrome P450 gene expression, down‐regulation of Nr4a, and greater inflammatory pathway activation. Very few exposure‐related responses occurred at any time in C57BL/6‐KO or ‐TG mice indicating that neither the short term nor long term responses of styrene in mice are relevant endpoints for assessing human risks. HIGHLIGHTSC57BL/6 KO & TG mice show few exposure related response at any time point.WT & CD‐1 show similar cellular pathway enrichment but differing in time course.WT mice respond with a single day; CD‐1 mice respond after several days of exposure.Results indicate non‐genotoxic mouse strain specific mode of action for short term exposure.Neither short nor long term responses of styrene in mice are relevant for human risk assessment.

Based on an analysis of mode of action, styrene-induced mouse lung tumors are not a human cancer concern.

ABSTRACT Based on 13 chronic studies, styrene exposure causes lung tumors in mice, but no tumor increases in other organs in mice or rats. Extensive research into the mode of action demonstrates the key events and human relevance. Key events are: metabolism of styrene by CYP2F2 in mouse lung club cells to ring‐oxidized metabolites; changes in gene expression for metabolism of lipids and lipoproteins, cell cycle and mitotic M‐M/G1 phases; cytotoxicity and mitogenesis in club cells; and progression to preneoplastic/neoplastic lesions in lung. Although styrene‐7,8‐oxide (SO) is a common genotoxic styrene metabolite in in vitro studies, the data clearly demonstrate that SO is not the proximate toxicant and that styrene does not induce a genotoxic mode of action. Based on complete attenuation of styrene short‐term and chronic toxicity in CYP2F2 knockout mice and similar attenuation in CYP2F1 (humanized) transgenic mice, limited metabolism of styrene in human lung by CYP2F1, 2 + orders of magnitude lower SO levels in human lung compared to mouse lung, and lack of styrene‐related increase in lung cancer in humans, styrene does not present a risk of cancer to humans. HighlightsThe mode of action for styrene‐induced mouse lung tumors is evaluated using the mode of action framework.Metabolism of styrene is catalyzed by CYP2F2 to ring‐oxidized metabolites in mouse lung.Metabolites cause changes in gene expression (circadian rhythm, cell cycle, nuclear transcription factors), not genotoxicity.Alterated processes lead to mitogenesis and cytotoxicity in bronchioles and eventually to preneoplastic/neoplastic lesions.There are no reactions in mouse lung to styrene oxide in the absence of further metabolism by CYP2F2.