TUMORICIDAL CAPACITIES OF MACROPHAGES PHOTODYNAMICALLY ACTIVATED WITH HEMATOPORPHYRIN DERIVATIVE

Four days after administration to mice of small amounts (30-600 ng/mouse) of hematoporphyrin derivative (HPD), peritoneal macrophages exhibited a greatly enhanced Fc-receptor mediated phagocytic capacity as assayed by ingestion activity of IgG-coated sheep erythrocytes. Much higher doses (greater than 3000 ng/mouse) did not have this effect. The peritoneal macrophages activated by administration of HPD have tumoricidal capacity for IgG-coated retinoblastoma cells. We then studied in vitro photodynamic activation of macrophages by white and red fluorescent light irradiation of mouse peritoneal cells (mixture of macrophages and B and T lymphocytes) in media containing very low concentrations of HPD. A short (5 s) white fluorescent light exposure (1Wm-2) of peritoneal cells in a medium containing 0.03 ng HPD/mL produced the maximal level of ingestion activity of macrophages. A 15 s red fluorescent light exposure (1Wm-2) of peritoneal cells in a medium containing 0.1 ng HPD/mL produced the maximal level of ingestion activity of macrophages. Thus, photodynamic activation of macrophages with white fluorescent light is more efficient than that with red fluorescent light. This can be explained by the fact that HPD has a large absorption peak at about 364 nm which extends into the visible range, and decreasingly smaller absorption bands at 500, 535, 570 and 630 nm. In vitro photodynamically activated macrophages showed efficient tumoricidal activity regardless of the type (white or red) of light used. These results suggest that a low level of HPD promotes therapeutic immunopotentiation.     

Activation of mouse macrophages by in vivo and in vitro treatment with a cyanine dye, lumin.

The cyanine photosensitizer, lumin, is a potent macrophage activating agent: 4 days after administration of small amounts of lumin to mice (20-40 ng mouse-1), peritoneal macrophages exhibited a greatly enhanced Fc-mediated ingestion activity; higher doses (more than 3000 ng mouse-1) did not have this effect. The in vitro photodynamic activation of macrophages in mouse peritoneal cells exposed to white fluorescent light (3 J m-2 s-1) was also studied in media containing various concentrations of lumin. A short light exposure (45 J m-2) with 10 ng lumin ml-1 produced a maximum ingestion activity of macrophages. Lumin has absorption peaks at 670 and 760 nm. Therefore we designed experiments in which peritoneal cells were exposed to a red fluorescent light (emission, 660 nm; 0.5 J m-2 s-1). In a medium containing 3 ng lumin ml-1 with 7.5 J m-2 of red light, a markedly enhanced ingestion activity of macrophages was observed. The photodynamic treatment of peritoneal macrophages alone did not stimulate phagocytic activity, but the photodynamic treatment of a mixture of non-adherent (B and T) cells and macrophages resulted in a greatly enhanced ingestion activity of macrophages. Thus non-adherent cells are required for the photodynamic activation of macrophages, implying that an activating factor is generated within the non-adherent cells and transmitted to the macrophages. This hypothesis was confirmed by the observation that co-cultivation of photodynamically treated non-adherent cells with untreated macrophages resulted in a greatly enhanced ingestion capacity.     

SENSITIVITY OF ACTIVATED MURINE PERITONEAL MACROPHAGES TO PHOTODYNAMIC KILLING WITH BENZOPORPHYRIN DERIVATIVE

Abstract— This study compared the ability of highly purified resting and activated DBA/2 mouse peritoneal macrophages to survive treatment with the photosensitizer benzoporphyrin derivative (BPD, verteporfin) and light. Culture of macrophages with recombinant murine interferon-γ (rIFN-γ, 100 U/mL) for 72 h imparted a phenotypic and functional activation by dramatically increasing cell surface expression of major histocompatibility complex Class II (Ia) molecules and the formation of nitric oxide. The rIFN-γ-activated macrophages were significantly (P < 0.05) more sensitive (lethal dose to cause a 50% reduction in cell survival, LD₅₀= 14.4 ± 1.1 ng/mL) to photodynamic killing with BPD and light (10 J/cm²) than cells (LD₅₀= 18.2 ± 2.0 ng/mL) cultured in medium alone. In contrast, macrophages treated with different concentrations of bacterial lipopolysaccharide (LPS) were as resistant or more resistant to photodynamic killing than cells cultured in medium alone. No cytotoxic effect of BPD was detected in cultures containing the drug but protected from light. Comparable amounts of BPD were taken up in vitro by unactivated and rIFN-γ-activated macrophages, as detected by flow cytometric analysis. However, cells cultured with LPS (10 μg/mL) took up more BPD than macrophages cultured in medium alone or with rIFN-γ. The DBA/2 P815 mastocytoma cells took up greater amounts of the drug and were subsequently more vulnerable to treatment with BPD and light (LD₅₀= 6.9 ng/mL) than macrophages cultured under any condition. The explanation for the increased vulnerability of rIFN-γ-activated macrophages and the greater resistance of LPS-activated macrophages, relative to medium-cultured macrophages, to photodynamic killing with BPD is uncertain. However, the increased susceptibility of macrophages, activated with the immunomodulatory cytokine IFN-γ, to treatment with BPD and light might indicate how photodynamic therapy could interfere with the development of experimental autoimmune disease, conditions in which activated macrophages are known to be involved.     

5-Aminolevulinic acid-induced protoporphyrin-IX accumulation and associated phototoxicity in macrophages and oral cancer cell lines

Studies were carried out on 5-aminolevulinic acid (ALA)-induced protoporphyrin (PpIX) synthesis in mice peritoneal macrophages and two human oral squamous cell carcinoma (OSCC) cell lines NT8e and 4451. Cells were treated with 200 microg/ml ALA for 15 h and PpIX accumulation was monitored by spectrofluorometry and phototoxicity to red light (630+/-20 nm) was measured by MTT assay. PpIX accumulation was higher in macrophages as compared to OSCC cells under both normal serum concentration (10%) and conditions of serum depletion. The results on phototoxicity measurements correlated well with the levels of PpIX accumulation in both macrophages and cancer cells. While red light caused 20% phototoxicity in macrophages, no phototoxicity was seen in 4451 cells at 10% serum. Decrease in serum concentration to 5% and 1% led to higher phototoxicity corresponding to 40% and 70% in macrophages and 10% and 15% in 4451 cells. Similar results were obtained in NT8e cell line. Propidium iodide staining followed by fluorescence microscopic observations on photodynamically treated co-culture of murine or human macrophages and cancer cells showed selective damage to macrophages. These results suggest that in OSCC, macrophages would contribute more to tumor PpIX level than tumor cells themselves and PDT may lead to selective killing of macrophages at the site of treatment. Since macrophages are responsible for production and secretion of various tumor growth mediators, the effect of selective macrophage killing on the outcome of PDT would be significant.     

Intravenously administered (1----3)-beta-D-glucan, SSG, obtained from Sclerotinia sclerotiorum IFO 9395 augments murine peritoneal macrophage functions in vivo.

Effect of intravenously (i.v.) or intraperitoneally (i.p.) administered (1----3)-beta-D-glucan, SSG, obtained from Sclerotinia sclerotiorum IFO 9395 on the murine peritoneal macrophage (PM) functions were examined. A single i.v. administration of SSG increased the number of PMs at a dose of 250 micrograms/mouse, and the peak appeared 4 d after administration. However, no special change was observed on peritoneal exude cell (PEC) populations. These PMs showed augmented lysosomal enzyme activity and the peaks appeared in 2 phases, on days 2 and 10. In contrast, SSG administered i.p. (250 micrograms/mouse) increased the number of PMs and enhanced the lysosomal enzyme activity of PMs from day 4, and a broad peak appeared until days 8--12. The populations of PECs were also changed by i.p. injection of SSG. Additionally, SSG administered i.v. enhanced phagocytic activity, H2O2 production and interleukin 1 (IL-1) production, and the kinetics of the activation differed depending on the activities. These data suggest that the effects of SSG on macrophage functions are different depending on administration routes, and there are some different mechanisms in the activation of macrophages in vivo by SSG.     

Activation of macrophage tumoricidal activity by photodynamic treatment in vitro-indirect activation of macrophages by photodynamically killed tumor cells

Macrophages constitute a major part of natural tumor defense by their capacity to destroy selectively a broad range of tumor types upon specific activation. In the last couple of years, these cells have also been implicated as effector cells in the destruction of tumors by photodynamic therapy. In the present work, the potential role of macrophage-mediated tumor cytotoxicity after photodynamic treatment in vitro has been investigated with respect to photodynamic activation of macrophages for tumoricidal effector functions. Our data show that photodynamic treatment of highly pure murine bone-marrow-derived macrophages with the hematoporphyrin derivative Photosan-3 does not result in activation of these cells for cytotoxicity against YAC-1 tumor cells or secretion of tumor necrosis factor and nitric oxide, irrespective of co-stimulation with interferon-γ, a potent priming agent for macrophage antitumoral activity. On the contrary, treatment with higher photosensitizer doses is found to reduce markedly the viability of the macrophage effector cells. Thus, these results do not lend any support to the hypothesis of direct macrophage activation by photodynamic treatment. However, macrophages are found to be activated for tumoricidal effector functions indirectly by photodynamically killed tumor cells, in a way reminiscent of phagocytosis-inducing stimuli. It is thus suggested that recognition and phagocytosis of photodynamically destroyed tumor cells constitutes the major signal for local activation of macrophages in photodynamically treated tumor tissues, which may be crucial for final, specific eradication by the immune system of tumor cells surviving photodynamic treatment.     

EFFECT OF PHOTODYNAMIC THERAPY ON ANTITUMOR IMMUNE DEFENSES: COMPARISON OF THE PHOTOSENSITIZERS HEMATOPORPHYRIN DERIVATIVE AND CHLORO-ALUMINUM SULFONATED PHTHALOCYANINE

The effects of the two photosensitizers chloroaluminum sulfonated phthalocyanine (ClAlSPc) and hematoporphyrin derivative (HpD) on the functional activities of macrophages and natural killer (NK) cells, two immunocyte populations implicated in the control of tumor development and spread, have been investigated. Murine peritoneal macrophages treated in vivo with ClAlSPc or HpD at 10 mg/kg body weight showed no impairment of Fc-mediated phagocytic capacity and only minor disturbances of in vitro tumoricidal/tumoristatic function. The NK cell activity of splenocytes obtained from photosensitizer-treated mice, assayed 24 or 48 h after i.v. injection of ClAlSPc or HpD at 10 mg/kg was unaffected compared to controls. However significant inhibition of NK activity was observed when splenocytes obtained from mice with or without subcutaneous Colo 26 tumors, treated with ClAlSPc plus laser therapy (675 nm) were used as effector cells. The results show that impairment of some anti-tumor activity can be observed in phthalocyanine treated or phthalocyanine + laser-treated animals but this relatively minor impairment may augur well for the use of systemic phthalocyanine administration in photodynamic therapy.     

GENETIC TOXICOLOGY OF THE PHOTOSENSITIZATION OF CHINESE HAMSTER CELLS BY PHTHALOCYANINES

Chloroaluminum phthalocyanine (CAPC) was recently shown to photosensitize cell killing in culture and tumor destruction in vivo. Because this compound is potentially useful in the photodynamic therapy of cancer, its properties as a genotoxic agent were evaluated. Applying the technique of alkaline elution to study DNA integrity, it was found that CAPC could produce single-strand breaks in the DNA of Chinese hamster cells after exposure to white fluorescent light. At equicytotoxic doses, the number of DNA strand breaks produced by CAPC photosensitization was about three times lower than that induced by X-irradiation. During incubation in growth medium after exposure to CAPC-plus-fluorescent light, cells rejoined DNA strand breaks at a rate similar to that observed after X-irradiation. Resistance to 6-thioguanine (6-TG') or to ouabain (OUA') were used as end points of mutagenic potential. Following a treatment that caused -90% cell killing, there was a slight mutagenic effect, i.e. the frequencies were increased by -40% above the background or spontaneous mutations. However, this enhancement was not statistically significant. Taken together, the foregoing, plus an earlier observation that there is no variation in the sensitivity of cells to CAPC + light through the cell cycle, lead to the inferences that DNA damage does not play a major role in cell killing and that the mutagenic potential of this treatment is small.     

Carbon dots with two-photon fluorescence imaging for efficient synergistic trimodal therapy

Applying the fluorescent carbon dots as smart materials in anticancer therapy is of great interest. However, carbon dots for multimodal synergistic anticancer therapy, especially for the triple modality, is rarely reported. Herein, we successfully synthesized OCDs by citric acid and(1R,2S)-2-amino-1,2-diphenylethan-1-ol, which show aggregation-induced emission property and two-photon fluorescence imaging. Meanwhile, OCDs are ideal photosensitizers for photothermal therapy under 808 nm and Type Ⅰ...     

ULTRAVIOLET RADIATION INDUCED CHANGES IN MACROPHAGE ACTIVITY

Abstract— The effect of ultraviolet (UV) radiation on macrophage activity was examined. Thioglycollate-stimulated peritoneal exudate cells were collected from adult C57BL/6 mice. Ninety-five per cent of the cells adhering to plastic petri dishes were macrophages as determined by the presence of a non-specific esterase. Adherent cells were exposed to UV radiation of 0.5-13.2 J/m². Viability and phagocytosis were measured at 0, 24, 48, 72 and 96 h after exposure. A statistically significant UV exposure-dependent decrease in macrophage viability and phagocytic capacity was observed. Macrophage viability and phagocytosis also decreased as a function of time after exposure to UV radiation.     

THE PHOTODEGRADATION OF PORPHYRINS IN CELLS CAN BE USED TO ESTIMATE THE LIFETIME OF SINGLET OXYGEN

NHIK 3025 cells were incubated with Photofrin II (PII) and/or tetra (3-hydroxyphenyl)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i.e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 3-6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PII present in the same cells. The distance diffused by the reactive intermediate, supposedly mainly 1O2, causing the photodegradation was estimated to be on the order of 0.01-0.02 micron, which corresponds to a lifetime of 0.01-0.04 microsecond of the intermediate in the cells. PII has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PII under the present conditions. Photoproducts (1O2 etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.     

DEPOLARIZATION OF MOUSE MYELOMA CELL MEMBRANES DURING PHOTODYNAMIC ACTION

The effect of photodynamic action on plasma membranes was examined using a fluorescent potentiometric indicator [di-SBA-C2(3)] to measure alterations in the plasma membrane potential of mouse myeloma cells treated with zinc phthalocyanine sulfonate and light. Plasma membrane depolarization was observed to be an early event in photodynamic action, showing both photosensitizer concentration and light dose dependence. Depolarization occurred while membrane integrity was retained and appears to be an early event preceding cell death.     

Effect of Photodynamic Pretreatment on the Susceptibility of Murine Tumor Cells To Macrophage Antitumor Mechanisms

Abstract— In vitro photodynamic treatment of YAC-1 murine T-lymphoma cells with the hematoporphyrin derivative Photosan 3 and red light resulted in dose-dependent phototoxicity. Photodynamic pretreatment, however, did not render these cells more susceptible to macrophage-me-diated tumor cytotoxicity or the cytotoxic effects of mac-rophage-derived antitumor mediators like tumor necrosis factor aL (TNF-aL) or interferon bT (IFN-bT). Independent of the degree of photosensitization used, the cytotoxicity values obtained with macrophages or the different mediators were shifted by the respective values for phototoxicity, suggesting these effects to be additive and thus not interdependent. These data show that while higher overall tumor cytotoxicity can be achieved by a combination of photodynamic treatment and macro-phage-mediated tumor destruction, this apparently is not a result of enhanced sensitivity of photodynamically treated tumor cells to macrophage antitumor mechanisms in general     

PHOTOSENSITIZED INACTIVATION OF CHINESE HAMSTER CELLS BY PHTHALOCYANINES

Chloroaluminum phthalocyanine was found to sensitize cultured Chinese hamster cells upon exposure to white fluorescent light. Elimination of wavelengths below 370 nm did not reduce the effect significantly, indicating that the effective wavelengths were those absorbed by the Q band (600–700 nm) of phthalocyanine. The magnitude of the photosensitizing effect increased with the dye concentration and the time of its contact with the cells prior to light exposure. Although photosensitization was drastically reduced in the absence of oxygen, the lack of effect of glycerol and D₂0 during exposure suggests that neither hydroxyl radicals nor ¹O₂ are responsible for the cytotoxic response. The efficiency of the photosensitized induced cell killing did not vary with the position of the cells in the cell cycle, in contrast to exposure to X-rays. The improved spectral properties, the reported low toxicity and the selective retention by neoplasms, make phthalocyanines promising candidates for use in photodynamic therapy of cancer.     

Sub-lethal Photodynamic Damage to ARPE-19 Cells Transiently Inhibits Their Phagocytic Activity†

Efficient phagocytosis of photoreceptor outer segments (POS) membranes by retinal pigment epithelium (RPE) plays a key role in biological renewal of these highly peroxidizable structures. Here, we tested whether photodynamic treatment, mediated by merocyanine 540 (MC 540), rose Bengal or a zinc-substituted chlorophyllide inhibited phagocytic activity of ARPE-19 cells in vitro. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS isolated from cow retinas and nonspecific phagocytosis of fluorescent polystyrene beads were measured by flow cytometry. Photodynamic treatment, mediated by all three photosensitizers with sub-threshold doses, induced significant inhibition of the cell-specific phagocytosis. The nonspecific phagocytosis was inhibited by photodynamic treatment mediated only by MC 540. The inhibition of phagocytosis was a reversible phenomenon and after 24 h, the photodynamically treated cells exhibited phagocytic activity that was comparable with that of untreated cells. This study provides proof of principle that sub-threshold photodynamic treatment of ARPE-19 cells with appropriate photosensitizers is a convenient experimental approach for in vitro study of the effects of oxidative stress on specific phagocytic activity of RPE cells. We postulate that oxidative damage to key components of the cell phagocytic machinery may be responsible for severe impairment of its activity, which can lead to retinal degeneration.     

Immune recognition of cytochrome c II. — The role of macrophages as modulators and not as antigen-presenting cells in immune response in vitro

C57BL mouse peritoneal macrophages sensitized with cytochrome c (cyt. c) in complete Freund adjuvant (CFA) in vivo were able to capture, internalize and reexpress horse cyt.c on their surface. The major portion of cyt.c captured came out of macrophages in 1 to 3 h, with molecular weight unmodified. Cyt.c could be partly cleaved into two fragments, with molecular masses of about 2-2.5 and 10 kD by external non-serine proteases. The dynamics of macrophage interactions with cyt.c coupled to fluorescent latex beads was also studied. Macrophages were shown not to present cyt.c in the immune response in vitro, but rather to modulate the response level. The activating substance was secreted into the culture fluid, while suppressive activity was mediated by the cells.     

Early effect of Mycobacterium tuberculosis infection on Mac-1 and ICAM-1 expression on mouse peritoneal macrophages

Effect of M. tuberculosis infection was studied on the expression of intercellular adhesion molocule-1 (ICAM-1) and Mac-1 markers on murine peritoneal macrophages. Intraperitoneal administration of M. tuberculosis resulted in a marked increase in the proportion of Mac-1(+) cells whereas the proportion of ICAM-1(+) cells declined sharply 4 h post infection. Absolute numbers of Mac-1(+) and ICAM-1(+) cells however increased at all time points after the infection. Comparison of kinetics of changes observed in Mac-1(+) and ICAM-1(+) cell populations with differential leukocyte counts in peritoneal cells indicated that these alterations could be due to cellular influx, especially that of neutrophils, or up regulation of these markers on macrophages and other peritoneal cells. In adherent peritoneal macrophages infected in vitro with M. tuberculosis, proportion of Mac-1(+) and ICAM-1(+) cells increased markedly within 24 h of infection. Mean expression of these markers on per cell basis also increased significantly. Similar results were obtained by using RAW 264.7 mouse macrophage cell line, suggesting that the enhanced expression of Mac-1 and ICAM-1 markers was a direct effect of M. tuberculosis infection and not mediated by contaminating cell types present in adherent macrophage preparations. Mac-1 and ICAM-1 expression was further studied on macrophages that had actually engulfed M. tuberculosis and compared with bystander macrophages without intracellular M. tuberculosis. For this purpose M. tuberculosis pre-stained with DilC18 fluorescent dye were used for infecting adherent peritoneal macrophages. Mac-1 and ICAM-1 expression on gated DilC18 positive and negative cell populations was analyzed. Our results indicate that the expression of Mac-1 and ICAM- 1 markers was significantly enhanced on all macrophages incubated with M. tuberculosis but was more pronounced on macrophages with internalized mycobacteria. Taken together, our results suggest that the expression of Mac-1 and ICAM-1 markers is significantly up regulated as a result of exposure and infection with M. tuberculosis. Since these markers play important role in the uptake of mycobacteria as well as in the process of antigen presentation by macrophages, their upregulation may be beneficial for generation of a protective immune response to M. tuberculosis.     

DNA LESIONS AND DNA DEGRADATION IN MOUSE LYMPHOMA L5178Y CELLS AFTER PHOTODYNAMIC TREATMENT SENSITIZED BY CHLOROALUMINUM PHTHALOCYANINE

Two closely related strains of mouse lymphoma L5178Y cells, LY-R and LY-S, have been found to differ in their sensitivity to the cytotoxic effects of photodynamic treatment (PDT) with chloroaluminum phthalocyanine (CAPC) and red light. Strain LY-R is more sensitive to photodynamic cell killing than strain LY-S. Differences in uptake of CAPC could not account for the differences in cytotoxic effects. There was no marked difference between the two strains in the induction of single-strand breaks (which includes frank single-strand breaks and alkali-labile lesions), but substantially more DNA-protein cross-links were formed in strain LY-R by CAPC and light. Repair of single-strand breaks proceeded with similar kinetics in both strains for the first 30 min post-irradiation, suggesting that these lesions are not responsible for the differential sensitivity of the two strains to the lethal effects of photodynamic treatment. Thereafter, alkaline elution revealed the presence of increasing DNA strand breakage in strain LY-R. DNA degradation, as measured by the conversion of prelabeled [14C] DNA to acid-soluble radioactivity, was more rapid and extensive in strain LY-R.     

Photodynamic activity of viral nanoparticles conjugated with C(60)

The development of viral nanoparticles (VNP) displaying multiple copies of the buckyball (C(60)) and their photodynamic activity is described. VNP-C(60) conjugates were assembled using click chemistry. Cell uptake and cell killing using white light therapy and a prostate cancer cell line is demonstrated.     

Laparoscopic photodynamic diagnosis of ovarian cancer peritoneal micro metastasis: an experimental study

The goal of this study was to assess the interest of photodynamic diagnosis (PDD) for laparoscopic detection of peritoneal micro metastasis in ovarian carcinoma. Using an experimental animal model, intraperitoneal injection of aminolevulinic acid (ALA) and hexylester of aminolevulinic acid (He-ALA) were compared in order to improve laparoscopic detection of ovarian peritoneal carcinomatosis. Twenty-one 344 Fischer female rats received an intra peritoneal injection of 106 NuTu-19 cells. At day 22, carcinomatosis with micro peritoneal metastasis was obtained. Rats were randomized in three groups concerning intra peritoneal injection before laparoscopic staging: 5-ALA hydrochloride, HE-ALA and sterile water. Using D Light system, laparoscopic peritoneal exploration was performed with white light (WL) first and then with blue light (BL). The main objective was to assess feasibility and sensibility of laparoscopic PDD for nonvisible peritoneal micro metastasis of ovarian cancer. The main parameter was the confirmation of neoplasic status of fluorescent foci by histology. Concerning PDD after intraperitoneal injection of 5-ALA, mean values of lesions seen is higher than without fluorescence (32 vs 20.7; P = 0.01). Using He-ALA, mean values of detected lesions is higher than without fluorescence (42.9 vs 33.6; P < 0.001). Neoplasic status of fluorescent foci was confirmed in 92.8% of cases (39/42). Using 5-ALA, fluorescence of cancerous tissue is significantly higher than that of normal tissue in all the rats (ratio 1.17) (P = 0.01). With He-ALA, intensity of fluorescence is significantly higher in cancerous tissue compared to normal tissue, irrespective of the rat studied (ratio 1.22; P < 0.001).