Increased dosage and treatment time of Epigallocatechin-3-gallate (EGCG) negatively affects skeletal parameters in normal mice and Down syndrome mouse models.

Raza Jamal,Joseph M Wallace,Matthew Blackwell,Kourtney Sloan,Roshni Patel,Jonathan Lacombe,Jared R Thomas,Randall J Roper,Stephen D Ginsberg

doi:10.1371/journal.pone.0264254

Abstract

Bone abnormalities affect all individuals with Down syndrome (DS) and are linked to abnormal expression of DYRK1A, a gene found in three copies in people with DS and Ts65Dn DS model mice. Previous work in Ts65Dn male mice demonstrated that both genetic normalization of Dyrk1a and treatment with ~9 mg/kg/day Epigallocatechin-3-gallate (EGCG), the main polyphenol found in green tea and putative DYRK1A inhibitor, improved some skeletal deficits. Because EGCG treatment improved mostly trabecular skeletal deficits, we hypothesized that increasing EGCG treatment dosage and length of administration would positively affect both trabecular and cortical bone in Ts65Dn mice. Treatment of individuals with DS with green tea extract (GTE) containing EGCG also showed some weight loss in individuals with DS, and we hypothesized that weights would be affected in Ts65Dn mice after EGCG treatment. Treatment with ~20 mg/kg/day EGCG for seven weeks showed no improvements in male Ts65Dn trabecular bone and only limited improvements in cortical measures. Comparing skeletal analyses after ~20mg/kg/day EGCG treatment with previously published treatments with ~9, 50, and 200 mg/kg/day EGCG showed that increased dosage and treatment time increased cortical structural deficits leading to weaker appendicular bones in male mice. Weight was not affected by treatment in mice, except for those given a high dose of EGCG by oral gavage. These data indicate that high doses of EGCG, similar to those reported in some treatment studies of DS and other disorders, may impair long bone structure and strength. Skeletal phenotypes should be monitored when high doses of EGCG are administered therapeutically.

Highlights

Trisomy 21 (Ts21) causes skeletal deficits in all individuals with Down syndrome (DS) that begin early in development, manifest in adolescence, and progress to osteoporosis
Treatment with 20 mg/kg EGCG for 7 weeks In Ts65Dn and littermate control male mice treated with ~20 mg/kg/day EGCG or vehicle, beginning at 3 weeks of age and lasting 7 weeks, trisomic mice displayed a genotypic effect with negative results in trabecular architecture compared with euploid mice (Table 2 and Fig 1)
From the study giving green tea extract (GTE) to individuals with DS, males with DS exhibited weight loss and females had a loss in bone mass [31]

Summary

Methods

AnimalsFemale B6EiC3Sn a/A-Ts(1716)65Dn (Ts65Dn—Stock #001924) and male B6C3F1 (Stock #100010) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). (Ts65Dn × B6C3F1) matings were continuously bred in our colony in rooms with a standard 12:12 light:dark cycle to generate and maintain the mice used in the study. New Ts65Dn and B6C3F1 mice from the Jackson Laboratory were added to the breeding colony every 6–12 months during all the experiments. Ts65Dn male mice are sterile, and because of the need to use female mice to maintain the breeding colony, only male mice were used in these studies. Male mice in the 20mg/ kg/day EGCG study were randomly selected from 27 litters with an average size of 4.9 total and 2.8 male mice (at postnatal day 10) per litter. Mice from these same litters were used for other studies and for colony propagation. Experiments with animals were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and received prior approval from the IUPUI School of Science Institutional Animal Care & Use Committee (IACUC) (SC213R and SC255R)

Results

Discussion

Conclusion