R123 Uptake Research Articles

Natural Furanocoumarin as Potential Oral Absorption Enhancer

Bioavailability of orally administered drugs can be influenced by many factors. Poor drug absorption across the intestinal membrane is one of the factors that contribute to low bioavailability of drugs. It has been suggested that the metabolism/active efflux in the small intestine is involved in the poor absorption of many drugs. Intestinal CYP3A4 and P-gp work coordinately to reduce the intracellular concentration of drugs. Recently, bioenhancers have been identified and extensively studied. The aim of this study was to evaluate natural furanocoumarins found in juices of common lime and kaffir lime as the potential enhancers for oral delivery by means of modulating CYP3A4 and/or P-gp activities. The role of isolated furanocoumarins on CYP3A4 was assessed by testosterone 6β-hydroxylation reaction, while the effect on P-gp was investigated using R123 and CAM uptake studies in Caco-2, as well as LLC-PK1and LLC-GA5-Col300. In the present study, we demonstrated that isopimpinellin isolated from common lime is the best CYP3A4 inhibitor among 4 isolated furanocoumarins, implying that isopimpinellin would possibly act as a bioenhancer by inhibiting pre-systemic metabolism. 6’,7’-Dihydroxybergamottin found in kaffir lime is a dual inhibitor of CYP3A4 and P-gp, suggest that it could potentially be used as a bioenhancer by inhibiting both pre-systemic metabolism and efflux mechanism. However,in vivostudy should be further conducted to confirm these effects in the body.

Mode of Action Analyses of Neferine, a Bisbenzylisoquinoline Alkaloid of Lotus (Nelumbo nucifera) against Multidrug-Resistant Tumor Cells.

Neferine, a bisbenzylisoquinoline alkaloid isolated from the green seed embryos of Lotus (Nelumbo nucifera Gaertn), has been previously shown to have various anti-cancer effects. In the present study, we evaluated the effect of neferine in terms of P-glycoprotein (P-gp) inhibition via in vitro cytotoxicity assays, R123 uptake assays in drug-resistant cancer cells, in silico molecular docking analysis on human P-gp and in silico absorption, distribution, metabolism, and excretion (ADME), quantitative structure activity relationships (QSAR) and toxicity analyses. Lipinski rule of five were mainly considered for the ADME evaluation and the preset descriptors including number of hydrogen bond donor, acceptor, hERG IC50, logp, logD were considered for the QSAR analyses. Neferine revealed higher toxicity toward paclitaxel- and doxorubicin-resistant breast, lung or colon cancer cells, implying collateral sensitivity of these cells toward neferine. Increased R123 uptake was observed in a comparable manner to the control P-gp inhibitor, verapamil. Molecular docking analyses revealed that neferine still interacts with P-gp, even if R123 was pre-bound. Bioinformatical ADME and toxicity analyses revealed that neferine possesses the druggability parameters with no predicted toxicity. In conclusion, neferine may allocate the P-gp drug-binding pocket and prevent R123 binding in agreement with P-gp inhibition experiments, where neferine increased R123 uptake.

P-glycoprotein mediated efflux limits substrate and drug uptake in a preclinical brain metastases of breast cancer model

The blood–brain barrier (BBB) is a specialized vascular interface that restricts the entry of many compounds into brain. This is accomplished through the sealing of vascular endothelial cells together with tight junction proteins to prevent paracellular diffusion. In addition, the BBB has a high degree of expression of numerous efflux transporters which actively extrude compounds back into blood. However, when a metastatic lesion develops in brain the vasculature is typically compromised with increases in passive permeability (blood-tumor barrier; BTB). What is not well documented is to what degree active efflux retains function at the BTB despite the changes observed in passive permeability. In addition, there have been previous reports documenting both increased and decreased expression of P-glycoprotein (P-gp) in lesion vasculature. Herein, we simultaneously administer a passive diffusion marker (14C-AIB) and a tracer subject to P-gp efflux (rhodamine 123) into a murine preclinical model of brain metastases of breast cancer. We observed that the metastatic lesions had similar expression (p > 0.05; n = 756–1214 vessels evaluated) at the BBB and the BTB. Moreover, tissue distribution of R123 was not significantly (p > 0.05) different between normal brain and the metastatic lesion. It is possible that the similar expression of P-gp on the BBB and the BTB contribute to this phenomenon. Additionally we observed P-gp expression at the metastatic cancer cells adjacent to the vasculature which may also contribute to reduced R123 uptake into the lesion. The data suggest that despite the disrupted integrity of the BTB, efflux mechanisms appear to be intact, and may be functionally comparable to the normal BBB. The BTB is a significant hurdle to delivering drugs to brain metastasis.

Mechanistic Studies of the Effect of Bile Salts on Rhodamine 123 Uptake into RBE4 Cells

To examine the ability of bile salts (BS) to act as permeation enhancers at the blood brain barrier, the effect of four BS (cholate, deoxycholate, monoketocholate and taurocholate) on accumulation of rhodamine 123 (R123) in rat brain endothelial (RBE4) cells was investigated. Experiments were performed using BS concentrations shown to be noncytotoxic to RBE4 cells. Uptake and efflux of R123 in the absence and presence of BS were studied by fluorescence spectroscopy and confocal microscopy. Changes in RBE4 cell membrane fluidity in the presence of BS were evaluated using fluorescence anisotropy. The direct interaction between BS and R123 (ion pairing) and the effect of BS on distribution of R123 into liposomes were studied by capillary electrophoresis. All BS influenced R123 uptake in a concentration-dependent manner and increased cell membrane fluidity. Monoketocholate produced the greatest increase in uptake and also significantly reduced R123 efflux probably by inhibition of P-glycoprotein (P-gp). Direct interaction of BS and R123 was weak, but distribution of R123 into liposomes was increased by BS. The results suggest that BS increase R123 uptake by increasing cell membrane fluidity and, in the case of MKC, by inhibiting P-gp.

Pgp inhibition by UIC2 antibody can be followed in vitro by using tumor-diagnostic radiotracers, 99mTc-MIBI and 18FDG

P-glycoprotein (Pgp, ABCB1) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing the phenomenon of multidrug resistance. It has been shown earlier that the combined application of a class of Pgp modulators (e.g. cyclosporine A and SDZ PSC 833) used at low concentrations and UIC2 antibody is a novel, specific, and effective way of blocking Pgp function ( Goda et al., 2007). In the present work we study the UIC2 antibody mediated Pgp inhibition in more detail measuring the accumulation of tumor diagnostic radiotracers, 2-[ 18F]fluoro-2-deoxy- d-glucose ( 18FDG) and [ 99mTc]hexakis-2-methoxybutyl isonitrile ( 99mTc-MIBI), into Pgp + (A2780AD) and Pgp − (A2780) human ovarian carcinoma cells. Co-incubation of cells with UIC2 and cyclosporine A (CSA, 2 μM) increased the binding of UIC2 more than 3-fold and reverted the rhodamine 123 (R123), daunorubicin (DNR) and 99mTc-MIBI accumulation of the Pgp + 2780AD cells to approx. the same level as observed in Pgp − cells. Similarly, 50 μM paclitaxel (Pacl) increased UIC2 binding, and consequently reinstated the uptake of R123, DNR and 99mTc-MIBI into the Pgp + cells. Blocking Pgp by combined treatments with CSA + UIC2 or Pacl + UIC2 also decreased the glucose metabolic rate of the A2780AD Pgp + cells measured in 18FDG accumulation experiments suggesting that the maintenance of Pgp activity requires a considerable amount of energy. Similar treatments of the A2780 Pgp − cells did not result in significant change in the R123, DNR, 99mTc-MIBI and 18FDG accumulation demonstrating that the above effects are Pgp-specific. Thus, combined treatment with the UIC2 antibody and Pgp modulators can completely block the function of Pgp in human ovarian carcinoma cells and this effect can be followed in vitro by using tumor-diagnostic radiotracers, 99mTc-MIBI and 18FDG.

Effect of Heat Preconditioning on the Uptake and Permeability of R123 in Brain Microvessel Endothelial Cells during Mild Heat Treatment

The purpose of this study was to assess the effect of mild heat and heat preconditioning on the uptake and permeability of a P‐glycoprotein (P‐gp) substrate, rhodamine 123 (R123), in a cell culture model of the blood–brain barrier (BBB). An immediate goal was to determine whether prior mild heat treatment could render brain microvessel endothelial cells more resistant to future heat stress and affect BBB drug permeation by future ultrasound‐induced mild heat (USMH) treatment. To address this issue, the expression level of two proteins, P‐gp and heat shock protein 70 (Hsp70), and their effects on uptake of R123 and permeability of R123 and [14C]‐sucrose in combination with mild heat and P‐gp modulator PSC833 during and after mild heat treatment in heat‐preconditioned and heat‐unconditioned bovine brain microvessel endothelial cell (BBMEC) monolayers were studied. Mild heat caused a significant increase in BBB permeability of R123 and [14C]‐sucrose when compared with control and PSC833. Exposure of BBMECs to heat preconditioning caused a slight but insignificant decrease in cellular uptake of R123 both during and immediately after mild heat treatment. Heat preconditioning also caused a slight but insignificant decrease in permeability of R123 and [14C]‐sucrose in BBMEC monolayers during mild heat treatment. Because exposure of BBMEC monolayers to mild heat did not affect P‐gp expression but slightly affected Hsp70 expression, a heat preconditioning that results in a reinforcement of the BBB other than increased expression of P‐gp is suggested. However, heat preconditioning is not sufficient to override the permeation‐enhancing effects of mild heat because mild heat caused a significant increase in R123 uptake and permeability of R123 and [14C]‐sucrose in both heat‐preconditioned and heat‐unconditioned cells. Because Hsp70 is known to play a major role in cellular repair and protective mechanisms, our results would imply a relative benign nature of mild heat treatment. Because heating produced by ultrasonic waves can be controlled and localized to a small volume within the tissue, the present results also suggest that USMH could play a pivotal role in the treatment of brain tumors and other brain‐related diseases.

The effects of cryopreservation on sperm morphology, motility and mitochondrial function.

The effects of cryoinjury were determined simultaneously on the mitochondrial function, motility, morphology and viability of ejaculated human sperm. Rhodamine 123 (R123) uptake (% of sperm) and stain intensity were used to determine sperm mitochondrial activity before and after cryopreservation from the semen of 50 men attending for infertility investigation. Morphology was assessed using Tygerberg's strict criteria and viability was assessed by eosin Y. Sperm motility was measured using computer-assisted semen analysis (CASA). Freeze-thawing caused a 37% (P = 0.001) reduction in normal morphological forms of sperm. All CASA sperm motility parameters except amplitude of lateral head displacement were similarly reduced. R123 uptake and intensity within sperm mitochondria decreased by 36 and 47% respectively (both P = 0.001). In addition, there was a similar significant decrease (31%, P = 0.001) in the viability of the sperm. Sperm morphology, motility, mitochondrial activities and viability are equally susceptible to cryopreservation-induced damage. R123 intensity is a novel and robust indicator of mitochondrial function before and after such trauma.

Overcoming MDR by Ultrasound-Induced Hyperthermia and P-Glycoprotein Modulation

We assessed the effects of combining ultrasound-induced hyperthermia (USHT) with the P-glycoprotein modulator PSC 833 on cellular retention and cytotoxicity of rhodamine 123 (R123) and doxorubicin in the parent and multidrug resistance (MDR) variants of two human cancer lines. USHT significantly increased cellular uptake of R123 and doxorubicin. Without PSC 833, release of R123 and doxorubicin from both USHT-treated and untreated cells was rapid. As expected, PSC 833 (0.5 μM) only slowed their release into the MDR lines. Interestingly, despite the differences in their starting amounts, PSC 833 was effective in prolonging R123 and doxorubicin release from both USHT-treated and untreated MDR cells. PSC 833 did not augment the cytotoxicity of doxorubicin in parent lines but did cause a significant increase in cytotoxicity of doxorubicin in the MDR lines. However, the combined effect of USHT and PSC 833 on cytotoxicity of doxorubicin far exceeded that produced by USHT or PSC 833 alone.

Influence of Multidrug Resistance (MDR) Proteins at the Blood–Brain Barrier on the Transport and Brain Distribution of Enaminone Anticonvulsants

Previous in vitro studies evaluating the permeability of enaminones suggested that their blood-brain barrier (BBB) transport might be influenced by the presence of an efflux mechanism. Therefore, transport mechanisms responsible for these anticonvulsants across the BBB were examined. The transport of enaminones (1 x 10(-4) M) were evaluated over 120 min with verapamil (50 microM) and probenecid (100 microM) using bovine brain microvessel endothelial cells (BBMECs) to assess the role of multidrug resistant (MDR) transport proteins [i.e., P-glycoprotein (Pgp) and MDR protein 1 (MRP1)] on efflux, respectively. Uptake studies in the presence and absence of rhodamine 123 (R123; 3.2 and 5.0 microM) were also performed in a Pgp overexpressing cell line, MCF-7/Adr. Select enaminone esters (12.5 mg/kg) were administered intravenously to mdr 1 a/b (+/+), mdr 1 a/b (-/-) knockout and probenecid pretreated mice (20 +/- 5g). Enaminones and R123 were assayed with validated ultraviolet and fluorescence high-performance liquid chromatography methods, respectively. Verapamil and probenecid significantly ( p>0.05) inhibited the transport of select enaminone esters across BBMECs. Two enaminones caused a statistically significant increase in the uptake of R123 in MCF-7/Adr cells. Concentrations of select enaminones in mdr 1 a/b (-/-) mice brains were significantly higher ( p<0.05) compared with those in mdr 1 a/b (+/+) mice brains; however, no differences were observed in probenecid pretreated animals. Taken together, these results strongly suggest that Pgp may influence enaminone transport at the BBB and hence affect epilepsy treatment with these agents.

HMG-CoA reductase inhibitors and P-glycoprotein modulation.

1. Five 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), (e.g. atorvastatin, fluvastatin, lovastatin, pravastatin and simvastatin), were investigated for their ability to reverse P-glycoprotein (P-gp) mediated rhodamine 123 (R123) transport in a murine monocytic leukaemia cell line that over-expresses the multi-drug resistance protein 1a/b (mdr1a/1b). 2. P-gp modulation was studied by a fluorimetric assay and confocal microscopy by means of R123 efflux and uptake experiments, respectively. 3. Atorvastatin acid, methyl ester and lactone, lovastatin lactone and simvastatin lactone inhibited R123 transport in a concentration-dependent manner. Lovastatin acid, simvastatin acid, fluvastatin and pravastatin did not show a significant inhibition of the R123 transport in our cell system. Atorvastatin methyl ester and lactone showed the highest affinities for P-gp and results were comparable for both methods. 4. In conclusion, monitoring of R123 transport in living cells by confocal microscopy in addition to fluorimetric assay is a sensitive tool to study P-gp affinity in drug screening that is especially useful for early phases of drug development.

Carrier-Mediated Uptake of Rhodamine 123: Implications on Its Use for MDR Research

We have examined the effects of verapamil and PSC 833 on cellular uptake and release of rhodamine 123 (R123) in two human cancer cell lines. Both verapamil and PSC 833 were able to increase R123 accumulation in the multidrug resistant (MDR) MV522/Q6 and KB-8-5 lines in the release study. However, the effects of these drugs on R123 accumulation during accumulation study were quite different. Incubation with PSC 833 increased R123 accumulation in both MDR lines. By contrast, incubation with verapamil only increased R123 accumulation in the KB-8-5 line. The failure of verapamil to increase R123 accumulation in the MV522/Q6 cells can be attributed to the presence of a carrier system in the parent MV522 cells that recognizes both R123 and verapamil, but not PSC 833, as substrates. These results imply that performing R123 accumulation study without first ascertaining possible role of a carrier system for cellular uptake of R123 and putative P-gp modulators might inadvertently lead one to draw improper conclusions on P-gp activity.

Disruption of mitochondrial activities in rabbit and human hepatocytes by a quinoxalinone anxiolytic and its carboxylic acid metabolite

The quinoxalinone anxiolytic, panadiplon, was dropped from clinical development due to unexpected hepatic toxicity in human volunteers. Subsequent experimental studies in rabbits demonstrated a hepatic toxicity that resembled Reye’s syndrome. In the present studies, we examined the effects of panadiplon and a metabolite, cyclopropane carboxylic acid (CPCA) on hepatic mitochondrial activities in vitro and ex vivo. Acute inhibition of β-oidation of [ 14C]palmitate was observed in rabbit and human hepatocyte suspensions incubated with 100 μM panadiplon. Panadiplon (30 μM) also reduced mitochondrial uptake of rhodamine 123 (R123) in cultured rabbit and human, but not rat hepatocytes, following 18 h exposure. CPCA also impaired β-oxidation and R123 uptake in rabbit and human hepatocytes. R123 uptake and β-oxidation in cells from some donors was not impaired by either agent, and cell death was not observed in any experiment. Hepatocytes isolated from panadiplon-treated rabbits had reduced palmitate β-oxidation rates and inhibited mitochondrial R123 uptake; R123 uptake remained inhibited until 48–72 h in culture. Rabbit mitochondrial respiration experiments revealed a slightly lower ratio of ATP formed/oxygen consumed in panadiplon-treated animals; direct exposure of normal rabbit liver mitochondria to panadiplon did not have this effect. Hepatocytes isolated from panadiplon-treated rabbits showed reduced respiratory control ratios and lower oxygen consumption compared to controls. Our results indicate that panadiplon induces a mitochondrial dysfunction in the liver, and suggest that this dysfunction may be attributed to the carboxylic acid metabolite.

Decreased mitochondrial function in quiescent cells isolated from multicellular tumor spheroids.

Cells in the inner region of multicellular spheroids markedly reduce their oxygen consumption rate, presumably in response to their stressful microenvironment. To determine the mechanism behind this metabolic adaptation, we have investigated relative mitochondrial mass and mitochondrial function in cells isolated from different regions of tumor spheroids by using a combination of mitochondrial-specific fluorescent stains and flow cytometric analysis. Uptake of rhodamine 123 (R123) is driven by the mitochondrial membrane potential and thus reflects mitochondrial activity. Uptake of 10-nonyl-acridine orange (NAO) reflects total mitochondrial mass independently of activity because this compound binds to cardiolipin in the inner mitochondrial membrane. NAO fluorescence per unit cell volume only decreased 10-20% for cells from the inner spheroid region compared with those near the surface. There was greater than a twofold reduction in R123 fluorescence in the inner region cells, however. Thus, tumor cells in spheroids alter their rate of respiration predominately by downregulating mitochondrial function as opposed to degradation of mitochondria. There was a correlation between R123 staining per unit cell volume and the growth fraction of the cells from spheroids, but not for monolayer cultures. We also show a linear correlation between R123 staining and the rate of oxygen consumption for both monolayer- and spheroid-derived cells. After separating the inner region cells from the spheroid and replating them in monolayer culture, the R123 uptake recovered to normal levels prior to entry of the cells into S-phase. This reduction in mitochondrial function in quiescent cells from spheroids can explain the long period required for these cells to re-enter the cell cycle and may have important implications for the regulation of tumor cell oxygenation in vivo.

Decreased mitochondrial function in quiescent cells isolated from multicellular tumor spheroids

Cells in the inner region of multicellular spheroids markedly reduce their oxygen consumption rate, presumably in response to their stressful microenvironment. To determine the mechanism behind this metabolic adaptation, we have investigated relative mitochondrial mass and mitochondrial function in cells isolated from different regions of tumor spheroids by using a combination of mitochondrial-specific fluorescent stains and flow cytometric analysis. Uptake of rhodamine 123 (R123) is driven by the mitochondrial membrane potential and thus reflects mitochondrial activity. Uptake of 10-nonyl-acridine orange (NAO) reflects total mitochondrial mass independently of activity because this compound binds to cardiolipin in the inner mitochondrial membrane. NAO fluorescence per unit cell volume only decreased 10–20% for cells from the inner spheroid region compared with those near the surface. There was greater than a twofold reduction in R123 fluorescence in the inner region cells, however. Thus, tumor cells in spheroids alter their rate of respiration predominately by downregulating mitochondrial function as opposed to degradation of mitochondria. There was a correlation between R123 staining per unit cell volume and the growth fraction of the cells from spheroids, but not for monolayer cultures. We also show a linear correlation between R123 staining and the rate of oxygen consumption for both monolayer- and spheroid-derived cells. After separating the inner region cells from the spheroid and replating them in monolayer culture, the R123 uptake recovered to normal levels prior to entry of the cells into S-phase. This reduction in mitochondrial function in quiescent cells from spheroids can explain the long period required for these cells to re-enter the cell cycle and may have important implications for the regulation of tumor cell oxygenation in vivo. J. Cell. Physiol. 176:138–149, 1998. Published 1998 Wiley-Liss, Inc.1 This article is a US Government work and, as such, is in the public domain in the United States of America.

Assessment of viability and mitochondrial function of equine spermatozoa using double staining and flow cytometry

An objective double-staining method was developed to evaluate viability and mitochondrial function of stallion spermatozoa using flow cytometry. Sperm viability was assessed by propidium iodide (PI) exclusion, and mitochondrial function was measured by the intensity of rhodamine 123 (R123) fluorescence. Flow cytometry estimates of sperm viability measured by PI were equivalent (P > 0.05) to estimates made using Hoechst 33258 stain and fluorescent microscopy (% dead: 25 ± 2.4 vs 21.5 ± 3.5). The use of both PI and R123 was validated by addition of various proportions of freeze-shocked (membrane damaged) cells to viable spermatozoa. There was a high correlation (r 2 = 0.996) between increased PI positivestained (dead) cells and the number of membrane-damaged spermatozoa added (% dead: 29 ± 0.4, 44 ± 1.4, 58 ± 0.9, 75 ± 0.7 and 91± 0.25 vs 0, 25, 50, 75 and 100% damaged cells, respectively). Optimal mitochondrial activity (OMA), as assessed by R123 uptake, was also reduced proportionally (r 2 = 0.976) by the percentage of membrane-damaged cells added (% OMA: 48 ± 0.6, 37 ± 1.7, 29 ± 0.5, 16 ± 1, 3.8 ± 1.3 vs 0, 25, 50, 75 and 100% damaged cells, respectively). The mitochondrial inhibitors rotenone and monensin significantly depressed optimal mitochondrial activity (P < 0.001), and there was a significant positive correlation (r 2 = 0.959) between the dose of inhibitors added and the population of sperm cells exhibiting minimal R123 staining (4 ∓ 0.9, 12 ∓ 1.6, 14 ∓ 0.1 and 28 ∓ 2% for treatments with 0, 0.5, 1 and 2 × 10 −5 M rotenone and 0, 0.5, 1, and 2 × 10 −4 M monensin, respectively). Finally, it was shown that treatments containing identical proportions of membrane-damaged cells yielded similar results in terms of viability and mitochondrial activity, irrespective of whether the staining procedure was single or double (P > 0.05). The results of the double-staining method revealed that the percentage of spermatozoa with optimally functioning mitochondria was significantly correlated with the percentage of viable (PI negative) sperm cells (r 2 = 0.998). Flow cytometric analyses using this staining procedure provides reliable and rapid (10,000 cells/min) qualitative assessment of stallion semen.

Mitochondrial injury to ram sperm during procedures associated with artificial insemination or frozen storage

The effect of a number of procedures involved in the preparation of ram semen for freezing or artificial insemination on sperm mitochondrial function were examined. Ram sperm mitochondrial function was assessed using the membrane potential probe rhodamine 123 (R123). Sperm R123 uptake was increased rather than reduced by incubation of sperm at a high rate of dilution (1: 30), and was unaffected by the addition of 1% serum albumin (BSA). Washing sperm also had no effect on R123 uptake. R123 uptake was reduced in ram sperm exposed to either hypertonic (450 mosmol l −1) or hypotonic (150 mosmol l −1) media ( P < 0.05) but was unaffected by varying pH from 5.9 to 8.9. There was no interaction between the effects of osmolality and pH. Mitochondrial injury due to cold shock became apparent when sperm were rapidly cooled below 10°C ( P < 0.05). Further damage occurred between 10 and 5°C ( P < 0.01). The effects of cold shock were not ameliorated by the addition of 1% BSA, 2 mM butylated hydroxytoluene (BHT) or 50 mM erythritol. The effect of freezing on ram sperm R123 uptake was greater than that of cold shock ( P < 0.01). R123 uptake after freezing and thawing in sperm treated with 250 mM sorbitol or 250 mM dimethylsulphoxide (DMSO) was greater than that of unprotected sperm, or sperm exposed to 4% glycerol ( P < 0.05). Accumulated R123 was released rapidly from frozen ram sperm ( P < 0.01) but released more slowly from cold shocked sperm ( P < 0.01). This suggests that the two stressors induce different types of mitochondrial injury.

Enhancement of CDDP Cytotoxicity by Caffeine Is Characterized by Apoptotic Cell Death

The ability of caffeine, an agent that suppresses cell replication by inhibiting deoxyribonucleic acids (DNA) repair, to modulate the cytotoxicity of cis -diamminedichloroplatinum (CDDP) was investigated in murine lymphoma cell line EL-4. EL-4 cells were precultured with or without 20 μg/ml CDDP for 1 h and then cultured in the presence of 5 mM caffeine up to 48 h after reseeding. In CDDP-pretreated cells, suppression of cell growth and decrease in cell viability from 24 h were observed. Cell cycle arrest in G2 + M phase and a concomitant increase in both rhodamine 123 (R123) uptake and cell size (forward scatter) were observed in these cells. Treatment with caffeine alone suppressed growth rate, R123 uptake, cell size, and frequency of S phase fraction in the cell cycle. Combination of the two agents, CDDP + caffeine, strongly suppressed not only cell viability but also R123 uptake and cell size, compared with CDDP pretreatment alone. DNA histogram analysis by flow cytometry revealed that cultivation with caffeine hastened G2 + M arrest in CDDP-pretreated cells by reduction in the time of passing through S phase. DNA fragmentation was observed following incubation of CDDP-pretreated cells with caffeine for 16 h when marked accumulation in G2 + M phase was observed. The intensity of these ladder fragments increased in a time-related manner. These results demonstrate that enhancement of cytotoxic activity against CDDP-treated cells by caffeine is characterized by an acceleration of DNA degradation in G2 + M phase, namely apoptotic cell death. The fact that induction of DNA fragmentation during G2 + M phase by caffeine modulates the cytotoxicity of CDDP may give rise to a new combination regime of chemotherapy against malignant tumor cells.

Assessment of P-glycoprotein-dependent drug transport in isolated rat hepatocytes using rhodamine 123

To test the activity of P-170 glycoprotein in isolated hepatocytes, a method has been developed employing the fluorescent dye rhodamine 123 (R-123). The uptake of R-123 by both freshly isolated and 4-hr-plated hepatocytes depends on dye concentration, time of incubation, and cell number. The efflux of R-123 from cells is inhibited by sodium azide and by verapamil. In standard conditions the efficiency of efflux of R-123 from cells correlates with the relative amount of immunoblottable glycoprotein. The method has been applied to detection of P-170 activity in hepatocytes from animals of different ages as well as from carcinogen-treated animals. The proposed assay appears a simple and adequate tool for the functional assessment of multidrug transporter in liver.

Increased uptake and retention of rhodamine 123 by mitochondria of old human fibroblasts

Binding of the fluorescent dye R123 by a variety of mammalian cells has been shown to be dependent on the high transmembrane potential maintained in functional mitochondria. Recent studies in our laboratory have shown that old human fibroblasts (HF) bind and retain more R123 than young HF. In an effort to determine whether this difference in R123 uptake indeed reflected a difference in mitochondrial transmembrane potential, drugs known to disrupt the transmembrane potential of mitochondria were used to monitor the R123-mitochondria interaction of young and old HF. Distinct differences indicating that old HF maintain a higher mitochondrial transmembrane potential were observed. More significantly, perhaps this difference reflects an age-related change(s) in the structure and/or function of mitochondria.

Staining of Plasmodium yoelii-infected mouse erythrocytes with the fluorescent dye rhodamine 123.

The cationic permeant fluorescent dye rhodamine 123 (R123) was used to stain Plasmodium yoelii-infected mouse erythrocytes. Fluorescence microscopic observations demonstrated that the parasite, but not the matrix of the infected erythrocyte, accumulated the dye. Differences in fluorescence intensity could not be found at the various developmental stages of the parasite; however, quantitation of the cell-associated dye revealed an increase in R123 uptake with parasite development. The retention of the parasite-associated dye, as measured by fluorescence microscopy and spectrophotometry after extraction of R123 with butanol, was markedly reduced by treatment of the infected erythrocytes with a proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and an inhibitor of proton ATPase, dicyclohexylcarbodiimide (DCCD). These results indicate that the accumulation and retention of R123 in P. yoelii reflect the parasite membrane potential and suggest that the parasite plasma membrane has a membrane potential-generating proton pump.