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Renal Tubular Pigmentation Associated with Senna-Related Metabolites

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“Renal Tubular Pigmentation Associated with Senna-Related Metabolites.” Willson GA (presenter) Malarkey DE, Allison N, Harris N, Miller RA. The 54th Annual Society of Toxicology Meeting. San Diego, CA. March 25, 2015.

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Renal Tubular Pigmentation Associated with Senna-Related Metabolites

  1. 1. Figure 17, 18 Martin, B.W., Terry, M.K, Bridges, C.H. and Bailey, E.M., Jr. (1981). Toxicity of Cassia occidentalis in the horse. Vet. Hum. Toxicol. Dec; 23(6):416-7 Mengs, U., Mitchell, J., McPherson, S., Gregson, R., and Tigner, J. (2004). A 13-week oral toxicity study of senna in the rat with an 8-week recovery period. Arch. Toxicol. 78:269-275. Mitchell, J.M., Mengs, U., McPherson, S., Zijlstra, J., Dettmar, P., Gregson, R., and Tigner, J.C. (2006). An oral carcinogenicity and toxicity study of senna (Tinnevelly senna fruits) in the rat. Arch. Toxicol. Oct. 5: 1-11. NTP. (2001). NTP toxicology and carcinogenesis studies of EMODIN (CAS No. 518-82-1) feed studies in F344/N rats and B6C3F1 mice. Natl Toxicol Program Tech Rep Ser. 493:1-278. NTP. (2005). NTP technical report on the toxicology and carcinogenesis studies of anthraquinone (CAS No.84-65-1) in F344/N rats and B6C3F1 mice (Feed Studies). Natl Toxicol Program Tech Rep Ser. 494: 1-358. Surh, I., Brix, A., French, J.E., Collins, B.J., Sanders, J.M., Vallant, M., and Dunnick, J. (2013). Toxicology and Carcinogenesis Study of Senna in C3B6.129F1-Trp53tm1Brd N12 Haplo- insufficient Mice. Toxicologic Pathology, 41: 770-778. Vashishtha, V.M., John, T.J. and Kumar, A. (2009). Clinical and pathological features of acute toxicity due to Cassia occidentalis in vertebrates. India J. Med. Res. Jul; 130(1): 23-30. Renal Tubular Pigmentation Associated with Senna-Related Metabolites Gabrielle A. Willson1, David E. Malarkey2, Neil Allison1, Nancy Harris1, and Rodney A. Miller1 1Experimental Pathology Laboratories, Inc. P.O. Box 12766, RTP, NC 27709 2Cellular and Molecular Pathology Branch, National Toxicology Program, National Institute of Environmental Health Sciences, P.O. Box 12233, RTP, NC 27709 Figure 5, 6 Introduction: Past oral administration of powdered senna to rats has resulted in unidentified pigment deposits in renal tubules (Mengs, et. al., 2004, Mitchell, et. al., 2006). Renal tubule brownish pigment was seen in mice and rats dosed with emodin (an anthraquinone metabolite of sennosides) and with anthraquinone in NTP studies. Since some species of Cassia (Senna) cause skeletal muscle degeneration and necrosis in domestic animals (but no reported renal pigment), we wanted see if the renal pigment in rodent studies was myoglobin or some other pigment(s) (Vashishtha, et.al., 2009, Martin, et. al, 1981). Methods: Slides from archived paraffin blocks from 4 rats and 4 mice with kidney pigment from the emodin and anthraquinone chronic studies were stained with Schmorl's, PAS, Hall's Bile, Prussian Blue stains and myoglobin by immunohistochemistry (IHC). Results: The IHC reaction for myoglobin resulted in positive staining of small intracellular granules of purple pigment within the proximal renal tubule epithelial cells in rat kidneys treated with emodin and anthraquinone. The same reaction did not stain any of the pigment as myoglobin in the mouse kidney sections. The Schmorl’s reaction was variably positive in rats. However, when positive, the reaction was associated with larger droplets and not the small distinct granules. The Prussian Blue reaction was variably positive in emodin rats and mice and anthraquinone rats. Anthraquinone mice were strongly positive for the iron stain. The PAS reaction and Hall’s bile stain were all negative for staining the small granules. Conclusion: It appears that a portion of the renal cortical tubule pigment in rats treated with emodin or anthraquinone may be myoglobin, whereas, in mice it does not. Lipofuscin was also a component of some of the brownish pigmentation, but was associated with larger droplets, not smaller distinct granule. This “wear and tear” pigment is likely related to the renal pathology induced by anthraquinone in rats and emodin treated rats and mice. PAS and Halls’ Bile stains were negative. In mice the only stain that was strongly positive was the Prussian Blue (iron) correlating with hemosiderin (not myoglobin) related to an induced anemia by anthraquinone in mice. The low numbers of animals limited group or sex comparisons and restricted evaluation to individual animal pigment staining results. Myoglobin positive and negative controls are shown (Figures 1 and 2). The brown pigment in emodin rats was mainly small intracytoplasmic granules in proximal tubules (Figure 3). Some brown pigment was seen interstitially and luminally, where it was less granular and more amorphous and “dusty”. The brown pigment in Emodin mice was less intracytoplasmic than in the rat and more luminal and interstitial with coarser clumps (Figure 5). It was less obvious than in the rat. The brown pigment in anthraquinone rats was mostly intracytoplasmic and present in small granules (similar to emodin rats) with some larger amorphous and homogeneous hyaline droplets which were also intracytoplasmic. In anthraquinone mice the pigment was intracytoplasmic and formed small golden brown granules (Figure 7). In the anthraquinone mice, the pigment was more uniformly distributed throughout the cortex than the other pigmented materials mentioned earlier. Results of Special Stains The immunohistochemical reaction for myoglobin resulted in positive (reddish-purple) staining of an estimated 25-40% of the distinct small intracellular granules of brown pigment seen only within the proximal renal tubule epithelial cells of all the rat kidney sections treated with emodin and anthraquinone (Figures 4 & 6). The same immunohistochemical reaction did not successfully identify any of the pigment as myoglobin in any of the mouse kidney sections. The Schmorl’s reaction (teal blue) for lipofuscin was somewhat positive for emodin rats (Figure 8) and positive for anthraquinone rats (Figure 10), whereas it was mostly negative for emodin mice (Figure 9) and negative for anthraquinone mice. However, when positive in rats, the positive reaction was associated with larger droplets and luminal smudgy pigment and not the small distinct granules. The PAS reaction (red) was mostly positive for larger droplets and negative for the small granules in emodin rats and anthraquinone rats (Figure 11 & 13). PAS stains were negative for mice (Figure 12 & 14). The Prussian Blue reaction (blue) in rats was mostly negative in emodin rats (Figure 15) and mice. Anthraquinone mice were strongly positive for the iron stain (Figure 18) and anthraquinone rats had mixed results, one mostly positive (Figure 16) and one mostly negative (Figure 17). Hall’s Bile reaction (green) was negative for all rats and mice. Figure 3, 4Results Reddish discoloration of the urine has been described as one of the side effects of senna laxative use in humans. In rats, oral administration of powdered senna for 13 weeks or for 104 weeks resulted in dark discoloration of the kidneys macroscopically and unidentified pigment deposits in renal tubules at the microscopic level (Mengs, et al. 2004; Mitchell, et al. 2006). No pigment was observed in the renal tubular epithelium of mice in a National Toxicology Program (NTP) study of Senna in the 39-Week Study of Senna in Heterozygous F1 p53 (+/-) Transgenic Mice. Renal tubule brownish pigmentation (unidentified) was reported in mice and rats dosed with emodin (an anthraquinone metabolite of sennosides) in a series of NTP studies ranging from 14 days to 2 years (NTP, 2001). Studies of anthraquinone have also reported increased incidences or severity of unidentified pigment deposits in the kidney of both rats and mice (NTP, 2005). Because Cassia (Senna) can produce skeletal muscle degeneration and necrosis in domestic animals, it was deemed interesting and useful to determine whether the renal pigment recorded in rodent studies is myoglobin or some other pigment(s). Introduction Figure 1, 2 Figure 7, 8 In this limited sampling of tissues, it appears that a portion of the renal cortical tubule pigment in rats treated with emodin or anthraquinone may be myoglobin, whereas, in mice it does not appear to be myoglobin. Lipofuscin also seemed to be a component (the less granular and more amorphous and “dusty”component) of some of the brownish pigmentation. This “wear and tear” pigment is likely related in some manner to the nephropathy induced by anthraquinone in rats and related to kidney changes of hyaline droplets (rats) and nephropathy (mice) in emodin treated rats and mice. PAS and Halls’ Bile stains did not contribute to definitive identification of the granular brown pigment. PAS-positive renal epithelial droplets were related to the anthraquinone induced renal lesions in rats and renal hyaline droplets induced by emodin in rats. In mice the only stain that was strongly positive was the Prussian Blue (iron) which indicated that the pigment in the renal cortical tubules was hemosiderin (not myoglobin) related to an induced anemia by anthra- quinone. The variable presence of Prussian Blue positivity in emodin-exposed rats and mice and anthraquinone exposed rats was likely related to renal pathology. The results of this probe into the origin of the renal tubule pigment indicate that the pigment may be the result of a myopathy in rats but not in mice. Discussion References To begin to address this issue we retrieved archived paraffin blocks and prepared kidney sections stained with Schmorl's, PAS, Hall's Bile, Prussian Blue stains and myoglobin by IHC (Novusbio, Lot # YF051410R) from the following rats and mice from the respective chronic studies: Methods Abstract Figure 11, 12 Figure 9, 10 Figure 13, 14 Emodin High dose male rat Emodin High dose female rat Emodin Mid dose male mouse Emodin High dose female mouse Anthraquinone High dose male rat Anthraquinone High dose female rat Anthraquinone Two high dose male mice Myoglobin IHC (red-purple) Schmorl’s (teal blue) PAS (red) Prussian Blue (blue) Hall’s Bile Stain (green) HM181 Emodin Rat Positive 50% Positive Negative granules Positive droplets Negative Negative HF473 Emodin Rat Positive Equivocal Negative granules Positive droplets Negative Negative MM185 Emodin Mouse Negative Negative Negative Negative Negative HF477 Emodin Mouse Negative Negative Negative Negative Negative HM239 Anthraquinone Rat Positive Positive Negative granules Positive droplets Positive Mild Negative HF494 Anthraquinone Rat Positive Positive Negative granules Positive droplets Negative Negative HM191 Anthraquinone Mouse Negative Negative Negative Positive Marked Negative HM125 Anthraquinone Mouse Negative Negative Negative Positive Marked Negative Figure 3. Rat Kidney, Negative control, myoglobin, A75723, 40X Figure 4. Rat Kidney, myoglobin, A75725, 40X Figure 5. Mouse Kidney, myoglobin, A75726, 40X Figure 6. Rat Kidney, myoglobin, A75727, 40X Figure 7. Mouse Kidney, myoglobin, A75729, 40X Figure 8. Rat kidney, Schmorl’s, A75730, 40X Figure 9. Mouse, Schmorl’s, A75732, 40X Figure 10. Rat, Schmorl’s, A75734, 40X Figure 11. Rat, PAS, A75735, 40X Figure 12. Mouse, PAS, A75737, 40X Figure 13. Rat, PAS, A75738, 40X Figure 14. Mouse, PAS, A75739, 40X Figure 17. Rat, Prussian Blue, A75742, 40X Figure 18. Mouse, Prussian Blue, A75743, 40X Figure 15, 16 Figure 15. Rat, Prussian Blue, A75740, 40X Figure 16. Rat, Prussian Blue, A75741, 40X Figure 1. Rat Heart. Image A75722, 40X objective. Negative myoglobin control Figure 2. Rat Heart. Image A75721, 40X objective. Positive myoglobin control Figure 3. Emodin rat HM181. Image A75723, 40X objective. Negative control myoglobin. The brown pigment in Emodin rats was mainly small intracytoplasmic granules in proximal tubules. Rats also had some pigment which was seen interstitially and luminally, where it was less granular and more amorphous and “dusty”. Figure 4. Emodin rat HF473. Image A75725, 40X objective. Myoglobin positive. The immunohistochemical reaction for myoglobin resulted in positive (reddish- purple) staining of distinct small intracellular granules of brown pigment only within the proximal renal tubule epithelial cells of all the rat kidney sections treated with Emodin. Figure 5. Emodin mouse MM185. Image A75726, 40X objective. Myoglobin negative. In Emodin in mice the brown pigment was less intracytoplasmic and more luminal and interstitial in coarser clumps. Figure 6. Anthraquinone rat HM239. Image A75727, 40X objective. Myoglobin positive. The immunohistochemical reaction for myoglobin resulted in positive (reddish-purple) staining of distinct small intracellular granules of brown pigment only within the proximal renal tubule epithelial cells of all the rat kidney sections treated with anthraquinone. Figure 7. Anthraquinone mouse HM191. Image A75729, 40X objective. Myoglobin negative. In anthraquinone mice the pigment in was intracytoplasmic and formed small golden brown granules. It was more uniformly distributed throughout the cortex than the pigment in Emodin rats and mice and anthraquinone rats. Figure 8. Emodin rat HM181. Image A75730, 40X objective. Schmorl’s stain. The Schmorl’s reaction (teal blue) for lipofuscin, when positive, was associated with larger droplets and luminal smudgy pigment and not the small distinct granules. Figure 9. Emodin mouse MM185. Image A75732, 40X objective. Schmorl’s stain. The Schmorl’s reaction (teal blue) for lipofuscin, when positive, was associated with larger droplets, interstitial, and luminal smudgy pigment and not the small distinct granules. Figure 10. Anthraquinone rat HF494. Image A75734, 40X objective. Schmorl’s stain. The Schmorl’s reaction (teal blue) for lipofuscin, when positive, was associated with larger droplets and luminal smudgy pigment and not the small distinct granules. Figure 11. Emodin rat HM181. Image A75735, 40X objective. PAS stain. The PAS reaction (red) was positive for larger droplets and negative for small granules in Emodin rats. Figure 12. Emodin mouse MM185. Image A75737, 40X objective. PAS stain. Negative staining of the brown particles. Figure 13. Anthraquinone rat HM239. Image A75738, 40X objective. PAS stain. The PAS reaction (red) was positive for larger droplets and negative for small granules in Anthraquinone rats. Figure 14. Anthraquinone mouse HM191. Image A75739, 40X objective. PAS stain. Negative staining of the brown particles. Figure 15. Emodin rat HM181. Image A75740, 40X objective. Prussian Blue stain. Negative staining of brownish granules. Figure 16. Anthraquinone rat HM239. Image A75741, 40X objective. Prussian Blue stain. Mostly positive. Figure 17. Anthraquinone rat HF494. Image A75742, 40X objective. Prussian Blue stain. Negative for small brown granules. Figure 18. Anthraquinone mouse HM125. Image A75743, 40X objective. Prussian Blue stain. The Prussian Blue reaction (blue) in Anthraquinone mice was strongly positive for the iron stain and uniformly distributed intracytoplasmically throughout the renal cortical epithelium. Figure 1. Rat Heart, Negative control, myoglobin, A75722, 40X Figure 2. Rat Heart, Positive control, myoglobin, A75721, 40X This may indicate that there is a coexisting muscle lesion present in rats. Senna has been associated with muscle lesions in other species. Conclusion

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