Distribution of anticancer antibiotic daunomycin in the rat heart and kidney revealed by immunocytochemistry using monoclonal antibodies

Antimicrob Agents Chemother

Matsunaga

et al. 2009

Self Cite

Gentamicin (GM) is a widely used antibiotic but shows renal toxicity. We produced a serum against GM (anti-GM) conjugated to bovine serum albumin with N-(gamma-maleimidobutyryloxy)succinimide. The antiserum was monospecific for GM and did not cross-react with the analog streptomycin, tobramycin, kanamycin, or amikacin. The antiserum also detected glutaraldehyde-fixed GM, and this enabled us to develop an immunocytochemical method for detecting the uptake of GM in rat kidney. Twelve hours after a single intravenous administration of GM, immunocytochemistry revealed that GM accumulated in the S1, S2, and S3 segments of the proximal tubules, as well as in the distal tubules and collecting ducts. By 12 h after injection, the drug was detected in cytoplasmic granules of the proximal tubule cells. However, early (1 h) after injection, drug accumulation was detected in the microvilli of these cells. The distal tubules and collecting ducts contained scattered swollen cells, reminiscent of necrotic cells, in which both the nuclei and the cytoplasm reacted strongly with GM. No staining occurred in the kidneys of saline-injected control rats. These results agree with previous studies showing that GM is endocytosed in the proximal tubules and accumulates in lysosomes. Additionally, our results show that GM also accumulates in the distal tubules and collecting ducts. This was achieved by systematically varying the pretreatment conditions-an approach necessary for detecting GM in different subcellular compartments. This approach should be useful for accurately detecting the uptake and toxicity of the antibiotic in different tissues.Gentamicin (GM) is widely used as a bactericidal agent for the treatment of severe infections with gram-negative bacteria, such as Pseudomonas aeruginosa. Its antibacterial activity is due to an irreversible inhibition of bacterial protein synthesis. However, clinical use of aminoglycosides, such as GM, is limited by their ototoxicity and nephrotoxicity. The mechanisms behind the nephrotoxity have been investigated extensively, but how these drugs induce cellular malfunction and necrosis remains to be elucidated. Accurate localization of GM in cells and tissues can be expected to contribute to this effort. Previously, subcellular fractionation (27), micropuncture techniques (24), and autoradiography using radioactively labeled aminoglycosides (6, 26) have been used to study the mechanisms of nephrotoxity. Additionally, immunogold labeling methods have been used to study the subcellular distribution of aminoglycosides in proximal tubules of the nephron (1,2, 3,4,20). However, these studies have been limited to ultrastructural analysis of proximal tubule cells. Recently, we have successfully developed immunocytochemical (ICC) procedures for detecting the cellular uptake of other water-diffusible small-molecule drugs, such as daunomycin (DM) (14,15,22,23). These procedures make use of glutaraldehyde (GA)-fixed tissues, which undergo a series of pretreatments to unmask sites of accumulation of the drug...

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Light-Microscopic Immunocytochemistry for Gentamicin and Its Use for Studying Uptake of the Drug in Kidney

Fujiwara

Antimicrob Agents Chemother

Matsunaga

et al. 2009

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“…The immunogen was prepared according to our previous method for antidaunomycin (DM) serum using a heterobifunctional agent, N-(␥-maleimidobutyryloxy)succinimide (GMBS) (8)(9)(10)17). Briefly, VM HCl (12.5 mg, 8.4 mol) in 0.1 M phosphate buffer (pH 6.8) and 1.0 ml and 0.7 mg (2.5 mol) GMBS in 0.5 ml tetrahydrofuran were mixed, constantly stirred, and incubated at room temperature (RT) for 90 min, yielding a GMBS-acylated VM solution.…”

Section: Preparation Of Immunogen (Vm-gmbs-bsa Conjugate)mentioning

confidence: 99%

“…The spleen cells (2 ϫ 10 8 ) from the immunized mouse and 3 ϫ 10 7 myeloma cells (P3/NS-1) were fused with the help of polyethylene glycol according to our previous method (17). Cells suspended in hypoxanthine-aminopterin-thymidine (HAT) medium were plated out in 96-well tissue culture plates (Corning) at a density of 10 5 cells per well in which 10 5 feeder cells had been plated.…”

Section: Preparation Of Immunogen (Vm-gmbs-bsa Conjugate)mentioning

confidence: 99%

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Immunocytochemistry for Vancomycin Using a Monoclonal Antibody That Reveals Accumulation of the Drug in Rat Kidney and Liver

Fujiwara

Yoshizaki

Antimicrob Agents Chemother

et al. 2012

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bWe prepared monoclonal antibodies against N-(␥-maleimidobutyryloxy)succinimide-conjugated vancomycin (VM). The monoclonal antibody was specific for conjugated or free VM. The monoclonal antibody enabled us to develop an immunocytochemical method for detecting the uptake of VM in the rat kidney and liver. Three hours after a single intravenous (i.v.) injection of VM at the therapeutic dose, the immunocytochemistry revealed that VM accumulated in large amounts in both the S1 and S2 segments and in much smaller amounts in the S3 segment of the proximal tubules as well as in the distal tubules and collecting ducts. The drug was detected in the cytoplasm, cytoplasmic irregular granules, nuclei, and microvilli of the proximal tubule cells. The distal tubules and collecting ducts contained scattered swollen cells in which both the nuclei and cytoplasm were heavily immunostained. Twenty-four hours after injection, most of the swollen cells returned back to normal size and had somewhat decreased immunostaining. Also, significant amounts of VM remained accumulated for as long as 8 days postadministration. In the liver, similar drug accumulation was observed in the Kupffer cells and the endothelial cells of the hepatic sinusoids but not in the hepatocytes, suggesting that vancomycin cannot be eliminated via the liver. Immunoelectron microscopic studies demonstrated that in the collecting ducts, uptake of VM occurred exclusively in the lysosomes and cytoplasm of the principal cells and scarcely in the intercalated cells. Furthermore, double fluorescence staining using rats simultaneously administered with VM and gentamicin strongly suggests that both drugs colocalized in lysosomes in the proximal tubule cells of kidneys.

Improved immunocytochemical detection of daunomycin

Ohara

Larsson

et al. 2007

Histochem Cell Biol

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Improved immunocytochemical (ICC) detection of the anthracycline anticancer antibiotic daunomycin (DM) has been achieved by use of hydrogen peroxide oxidation prior to ICC staining for DM. The new method greatly enhanced the localization of DM accumulation in cardiac, smooth and skeletal muscle of rats after a single i.v. dose of the drug. DM accumulated in the nuclei as well as in the sarcoplasm, where it occurred in the form of small granules, which were particularly evident in cardiac muscle cells. The distribution of the granules coincided with that of mitochondria. Uptake of DM in nuclei and mitochondria of heart muscle cells may help to improve our understanding of the cardiac toxicity of DM and related anthracycline antibiotics. A number of ELISA tests were carried out in order to elucidate the mechanisms of H2O2-assisted antigen retrieval. A possible mechanism is that DM is reduced and converted to its semiquinone and/or hydroquinone derivative in vivo. Oxidation by hydrogen peroxide acts to convert these derivatives back to the native antigen. The improved ICC methodology using oxidation to recreate native antigens from reduced metabolites may be helpful also with respect to the localization of other drugs.