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Fluorescence imaging for a noninvasive in vivo toxicity-test using a transgenic silkworm expressing green fluorescent protein.

Inagaki Y, Matsumoto Y, Ishii M, Uchino K, Sezutsu H, Sekimizu K - Sci Rep (2015)

Bottom Line: The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells.In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body.Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 111-0033, Japan.

ABSTRACT
In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These findings suggest that the transparent GFP-expressing silkworm model is useful for evaluating the toxicity of chemicals that induce tissue injury.

No MeSH data available.


Related in: MedlinePlus

Anatomy of GFP transgenic silkworms.A, Bright field; B, Under excitation light; C, Dissection in brightfield; D, Dissection under excitation light. The left pictures of C and D show the dissected whole body after removal of the gut. The fat body was distributed under the epidermal tissue. The right pictures of C and D show isolated gut.
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f1: Anatomy of GFP transgenic silkworms.A, Bright field; B, Under excitation light; C, Dissection in brightfield; D, Dissection under excitation light. The left pictures of C and D show the dissected whole body after removal of the gut. The fat body was distributed under the epidermal tissue. The right pictures of C and D show isolated gut.

Mentions: Anatomical analysis of the transgenic GFP-expressing silkworms revealed that GFP was strongly expressed in the gut and fat body, which functions similarly to the mammalian liver (Fig. 1). Injection of carbon tetrachloride (CCl4) led to an increase in the fluorescence intensity of GFP in the hemolymph of the transgenic silkworm in a dose-dependent manner (Fig. 2). By contrast, no fluorescence was observed in the hemolymph of silkworms injected with olive oil, the carrier solvent for CCl4. This result suggests that tissue injury induced by CCl4 caused GFP to leak from the tissue cells into the hemolymph of the transgenic silkworm. We previously reported that injection of cytotoxic chemicals, including salicylic acid and iron sulfate, increases the activity of alanine-aminotransferase (ALT), a marker enzyme for liver injury in humans, in the silkworm hemolymph9. In the present study, we observed that the fluorescence intensity in the hemolymph of the transgenic silkworm increased following injection with these cytotoxic compounds in a dose-dependent manner (Fig. 3). These results suggest that tissue-injuring chemicals induced GFP to leak into the hemolymph from damaged tissues in transgenic silkworms.


Fluorescence imaging for a noninvasive in vivo toxicity-test using a transgenic silkworm expressing green fluorescent protein.

Inagaki Y, Matsumoto Y, Ishii M, Uchino K, Sezutsu H, Sekimizu K - Sci Rep (2015)

Anatomy of GFP transgenic silkworms.A, Bright field; B, Under excitation light; C, Dissection in brightfield; D, Dissection under excitation light. The left pictures of C and D show the dissected whole body after removal of the gut. The fat body was distributed under the epidermal tissue. The right pictures of C and D show isolated gut.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4462092&req=5

f1: Anatomy of GFP transgenic silkworms.A, Bright field; B, Under excitation light; C, Dissection in brightfield; D, Dissection under excitation light. The left pictures of C and D show the dissected whole body after removal of the gut. The fat body was distributed under the epidermal tissue. The right pictures of C and D show isolated gut.
Mentions: Anatomical analysis of the transgenic GFP-expressing silkworms revealed that GFP was strongly expressed in the gut and fat body, which functions similarly to the mammalian liver (Fig. 1). Injection of carbon tetrachloride (CCl4) led to an increase in the fluorescence intensity of GFP in the hemolymph of the transgenic silkworm in a dose-dependent manner (Fig. 2). By contrast, no fluorescence was observed in the hemolymph of silkworms injected with olive oil, the carrier solvent for CCl4. This result suggests that tissue injury induced by CCl4 caused GFP to leak from the tissue cells into the hemolymph of the transgenic silkworm. We previously reported that injection of cytotoxic chemicals, including salicylic acid and iron sulfate, increases the activity of alanine-aminotransferase (ALT), a marker enzyme for liver injury in humans, in the silkworm hemolymph9. In the present study, we observed that the fluorescence intensity in the hemolymph of the transgenic silkworm increased following injection with these cytotoxic compounds in a dose-dependent manner (Fig. 3). These results suggest that tissue-injuring chemicals induced GFP to leak into the hemolymph from damaged tissues in transgenic silkworms.

Bottom Line: The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells.In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body.Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 111-0033, Japan.

ABSTRACT
In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These findings suggest that the transparent GFP-expressing silkworm model is useful for evaluating the toxicity of chemicals that induce tissue injury.

No MeSH data available.


Related in: MedlinePlus