Effect of anaesthesia-induced alterations in haemodynamics on in vivo kinetics of nitroxyl probes in electron spin resonance spectroscopy

Although the advent of in vivo electron spin resonance (ESR) spectroscopy has allowed analysis of the redox status of living animals, whether the haemodynamic condition affects the signal decay rate remains unknown. Three kinds of haemodynamic conditions were generated by changing the anaesthetic dosage in mice. Haemodynamics was analysed (n=6 each) and in vivo ESR was performed to measure the signal decay rates of three nitroxyl spin probes (carbamoyl-, carboxy- and methoxycarbonyl-PROXYL) at the chest and head regions (n=6 for each condition and probe). Haemodynamic analysis revealed negative inotropic and chronotropic effects on the cardiovascular system depending on the depth of anaesthesia. Although signal decay rates differed among three probes, they were not affected by heart rate alteration. In this study we report the haemodynamics-independent signal decay rate of nitoxyl probes.     

Pharmacokinetic Analysis of Free Radicals by in vivo BCM (Blood Circulation Monitoring)-ESR Method

In pharmacokinetic studies, a variety of analytical method including radioisotopic detection and HPLC (high performance liquid chromatography) has been used. In the present investigation, we developed in vivo BCM (Blood Circulation Monitoring)-ESR method, which is a new technique with a conventional X-band ESR spectrometer for observing stable free radicals in the circulating blood of living rats under anaesthesia. Both 5-(PROXYL derivatives) and 6-(TEMPO derivatives) membered nitroxide spin probes with various types of substituent functional group were used. After physicochemical properties of the spin probes such as hyperfine coupling constant (A-value), g-value and partition coefficient as well as chemical stability of the compounds in the fresh blood were obtained, the in vivo BCM-ESR method was performed in normal rats. Several pharmacokinetic parameters such as half-life of the probes, distribution volume, total body clearance and mean residence time were obtained and discussed in terms of their chemical structures. In addition, clearance of a spin probe was related to the urine concentration. The BCM-ESR method was found to be very useful to observe free radicals at the real time. By time-dependent ESR signal decay of spin probes, pharmacokinetic parameters were obtained.     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Non-invasive analysis of reactive oxygen species generated in rats with water immersion restraint-induced gastric lesions using in vivo electron spin resonance spectroscopy

Reactive oxygen species (ROS) are reportedly associated with gastric ulcer. We previously reported the use of an in vivo 300-MHz electron spin resonance (ESR) spectroscopy/nitroxyl probe technique to detect _•OH generation in the stomachs of rats with gastric ulcers induced by NH₄OH. However, this is an acute ulcer model, and the relationship between in vivo ROS generation and lesion formation remains to be clarified. To address this question, the same technique was applied to a sub-acute water immersion restraint (WIR) model. A nitroxyl probe that was less membrane-permeable was orally administered to WIR-treated rats, and the spectra in the gastric region were obtained by in vivo ESR spectroscopy. The signal intensity of the orally administered probe was clearly changed in the WIR group, but no change occurred in the control group. Both enhanced signal decay and neutrophil infiltration into mucosa were observed 2 h after WIR with little formation of any mucosal lesions. The enhanced signal decay was caused by _•OH generation, based on the finding that the decay was suppressed by mannitol, desferrioxamine and catalase. Intravenous treatment with either anti-neutrophil antibody or allopurinol also suppressed the enhanced signal decay, and allopurinol depressed neutrophil infiltration into the mucosa. In rats treated with WIR for 6 h, lesion formation was suppressed by 50% with all antioxidants used in this experiment except anti-neutrophil antibody. These findings suggest that _•OH, which is generated in the stomach via the hypoxanthine/xanthine oxidase system upon neutrophil infiltrated into the mucosa, induces mucosal lesion formation, but that it accounts for only half the cause of lesion formation.     

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy in order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure and conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules (the increased h₊₁/h₀ parameter of maleimide spin label, MSL; 0.377 ± 0.021 in diabetics vs 0.338 ± 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (τ_c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 ± 0.42 in diabetics, n = 21 vs 4.79 ± 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 ± 1.08, n = 28 vs 7.31 ± 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h₊₁/h₀ parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.     

Spinnokinetic analyses of blood disposition and biliary excretion of nitric oxide (NO)-Fe(II)-N-(dithiocarboxy)sarcosine complex in rats: BCM-ESR and BEM-ESR studies

Nitric oxide (NO) is well known to have a wide variety of biological and physiological functions in animals. On the basis of the fact that Fe(II)-dithiocarbamates react with NO, a Fe(II)-N-(dithiocarboxy)sarcosine complex (Fe(II)-DTCS) was proposed as a trapping agent for endogenous NO. However, quantitative pharmacokinetic investigation for NO-Fe(II)-dithiocarbamate complexes in experimental animals has been quite limited. This paper describes the results on the quantitative pharmacokinetic features of a NO-Fe(II)-N-DTCS in both the blood and bile of rats following intravenous (i.v.) administration of the complex. For this purpose, we applied two in vivo methods, i.e. (1) in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) which previously developed, and (2) in vivo biliary excretion monitoring-electron spin resonance (BEM-ESR). We monitored real-time ESR signals due to nitrosyl-iron species in the circulating blood and bile flow. The ESR signal due to NO-Fe(II)-DTCS was stable in biological systems such as the fresh blood and bile. In in vivo BCM- and BEM-ESR, the pharmacokinetic parameters were calculated on the basis of the two-compartment and hepatobiliary transport models. The studies also revealed that the compound is widely distributed in the peripheral organs and partially excreted into the bile. We named a kinetic method to follow spin concentrations as spinnokinetics and this method will be useful for detecting and quantifying the endogenously generated NO in Fe(II)-DTCS administered animals.     

Transfer of nitric oxide from the liver to erythrocytes--an ESR study using nitroglycerin-treated mice

Nitric oxide (NO) formation in the liver and blood of the mouse following intraperitoneal treatment with nitroglycerin (glycerol trinitrate, GTN) was determined using electron spin resonance (ESR) spectroscopy. ESR signals of heme-NO complexes were detected at maximum levels within 5 min in the liver, but increased to a maximum level about 15-30 min later in the blood. GTN is not metabolized to release NO in vitro in the blood of the mouse. The hepatic microsomes which showed the heme-NO complexes ESR signals were incubated with mouse erythrocytes, with the result that a hemoglobin-NO signal was obtained from the erythrocytes. The activities of microsomal cytochrome P-450, the hepatic level of glutathione, and the reduction rate of nitroxide radicals in the in vivo liver, measured using L-band ESR spectroscopy, were temporarily decreased following GTN administration. In conclusion, NO in the liver could be scavenged by circulating erythrocytes, which might minimize NO-induced liver damage.     

Kinetic study on ESR signal decay of nitroxyl radicals,potent redox probes for in vivo ESR spectroscopy,caused by reactive oxygen species

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.     

In vivo L-band ESR and quantitative pharmacokinetic analysis of stable spin probes in rats and mice

Free radical species in animals have been measured by X-band ESR spectrometric method on a block of organs or a portion of homogenized samples. However, a nondestructive in vivo ESR measurement has been realized by using a recently developed L-band ESR spectrometry. With this L-band ESR method, we measured ESR spectra in animals, who received stable nitroxide radicals. L-band ESR spectra were observed at the upper abdomen of mice as well as at the heads of mice and rats at various ages immediately after the intravenous injections of nitroxide radicals such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (3-carbamoyl-PROXYL), in which ESR measurements of the radicals were performed noninvasively at the real time. On the basis of the observed time-dependent free radical clearance curves, the following important results were obtained: (1) Free radical clearances were able to analyze by the pharmacokinetic method. (2) The radicals at the head of mice, given 4-hydroxy-TEMPO, were determined quantitatively by a new analytical method using L-band ESR for the first time. (3) The elimination of the radical was found to be saturated in mice. (4) The clearance rate constant of 4-hydroxy-TEMPO detected at the head of mice was decreased in dose- and age-dependent manners. While, no age-dependent clearance rate constant of 4-hydroxy-TEMPO was observed at the upper abdomen of mice. (5) Ratios of the amount of the detected radicals to that of the administered radicals were decreased age-dependently, but they were independent of the dose of the radicals, suggesting the age-dependent decrease of distribution capacity ratio of the radical at the head of animals. (6) Clearance rate constants of 4-hydroxy-TEMPO and 3-carbamoyl-PROXYL, that were estimated by X- and L-band ESR for the collected blood of mice and rats, were found to be remarkably smaller than those in whole living animals observed by in vivo L-band ESR method. The results suggest that the clearance of the nitroxide radical is relevant to the alteration of the radical in animals following the change of organ distribution and metabolism. (7) Both the radical and its corresponding hydroxylamine, which is the reduced form of the radical, were detectable by X-band ESR method in the collected urine of mice and rats without and with an oxidizing agent, respectively.

On the basis of the results on L-band ESR spectrometry, the first quantitative pharmacokinetic analysis of stable spin probes in animals is proposed.     

不同刺激方式对在体和离体蛙腓肠肌单收缩影响的定量分析*

目的: 自编程定量分析不同电刺激方式对在体和离体蛙腓肠肌单收缩的影响。方法: 实验分为四个组:间接刺激在体标本组(n=12),直接刺激在体标本组(n=8),间接刺激离体标本组(n=12),直接刺激离体标本组(n=8)。分别制备在体和离体蛙腓肠肌标本, 施以间接电刺激(经坐骨神经)或直接电刺激(细针灸针刺激电极直接刺入腓肠肌中):强度从0 V开始,周期3 s,增量0.02 V,刺激150次;用生物机能实验系统(BL-420F)实时记录不同强度刺激对肌肉收缩的影响。后续通过自编程辅助处理分析肌肉收缩数据,对单收缩特征参数进行定量比较分析。 结果: ①对在体标本,与直接刺激比较:间接刺激的阈强度、半高强度和最适强度均更小(P＜0.05);最大单收缩幅度更大,收缩期更长,上升斜率更小(P＜0.05)。②对离体标本,与直接刺激比较:间接刺激的阈强度、半高强度和最适强度也均更小(P＜0.05);最大单收缩幅度更大,收缩期更长,上升斜率更小(P＜0.05)。③均采用间接刺激或直接刺激时,与离体标本比较,在体标本单收缩的各项参数均无差异(P＞0.05)。 结论: 不论使用间接刺激或是直接刺激,在体标本和离体标本的单收缩功能特点均无显著差异; 但间接刺激比直接刺激更容易触发腓肠肌产生单收缩,且单收缩幅度更高。     

In Vivo Esr Studies of Antioxidant Activity on Free Radical Reaction in Living Mice Under Oxidative Stress

In vivo antioxidant activity seems to be quite complicate due to multiple interaction with biomaterials and differs from results by in vitro experiments. In vivo estimation of antioxidant activity is performed by measuring TBA reactive substances in blood or hydrocarbon gases in breath, but these systems do not measure free radical reaction but the final products of oxidative reaction. In the present study, we applied in vivo ESR to evaluate antioxidant activity by monitoring the redox reaction of nitroxide radical and clearly found that the nitroxide is very susceptible to oxidative stress in vivo and quite useful to evaluate antioxidant activity non-invasively.     

Confirmation of superoxide generation via xanthine oxidase in streptozotocin-induced diabetic mice

Reactive oxygen species (ROS) may play key roles in vascular inflammation and atherogenesis in patients with diabetes. In this study, xanthine oxidase (XO) system was examined as a potential source of superoxide in mice with streptozotocin (STZ)-induced experimental diabetes. Plasma XO activity increased 3-fold in diabetic mice (50±33 μU/ml) 2 weeks after the onset of diabetes, as compared with non-diabetic control mice (15±6 μU/ml). In vivo superoxide generation in diabetic mice was evaluated by an in vivo electron spin resonance (ESR)/spin probe method. Superoxide generation was significantly enhanced in diabetic mice, and the enhancement was restored by the administration of superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), which was reported to scavenge superoxide. Pretreatment of diabetic mice with XO inhibitors, allopurinol and its active metabolite oxipurinol, normalized the increased superoxide generation. In addition, there was a correlation (r=0.78) between the level of plasma XO activity and the relative degree of superoxide generation in diabetic and non-diabetic mice. Hence, the results of this study strongly suggest that superoxide should be generated through the increased XO seen in the diabetic model mice, which may be involved in the pathogenesis of diabetic vascular complications.     

Pasteur effect in the in vitro vascularly perfused rat small intestine

Isolated small intestine perfused in vitro with media with low oxygen concentration was found to contain low levels of ATP when compared with rat small intestine in vivo. The addition of fluorocarbon FC 75 to an erythrocyte-free perfusion medium was found to result in a high phosphate potential and a low rate of lactate production from glucose in isolated perfused small intestine, resembling the in vivo condition. This allowed the demonstration of a Pasteur effect in that replacement of oxygen by nitrogen (or the addition of 2,4-dinitrophenol) led to a rapid increase of the rate of glycolysis, and a decrease of the ATP concentration in the tissue     

纳米卟啉金属有机骨架对斑马鱼神经系统发育的作用*

目的:研究纳米卟啉金属有机骨架(NPMOF)对斑马鱼幼鱼神经系统发育的作用。方法:斑马鱼胚胎在发育6 h(hpf)后随机分为两组:对照组(n=500)和暴露组(n=500),对照组斑马鱼孵化于E3溶液中,暴露组于100 mg/L的NPMOF-E3溶液中,持续暴露至28、48、72、96或120 hpf。以20条斑马鱼为一批,在28、48、72、96和120 hpf分别提取3批实验组和对照组的总RNA用于RT-PCR;在120 hpf,实验组和对照组分别取20条PTU处理过的斑马鱼用于整体胚胎原位杂交,同样是在120 hpf,每组取150条用于免疫荧光染色,30条用于行为学测试。NPMOF的形状和尺寸用透射电镜测定,其光学特性由紫外分光光度计和荧光光谱仪测定;通过免疫荧光、整体胚胎原位杂交和RT-PCR方法检测各类神经细胞的发育;行为学测试用来监测斑马鱼运动状态的改变。结果:与正常组比较,NPMOF暴露组中神经发育相关基因的表达明显升高(P＜0.05),müller细胞和星形胶质细胞的数量明显增加(P＜0.05),神经元和少突胶质细胞的分布与形态未显示出差异;行为学测试中,斑马鱼的总运动距离、快速运动时间和运动速度的值显著增加(P＜0.05),总静止时间明显降低(P＜0.05)。结论:100 mg/L NPMOF溶液的持续暴露对斑马鱼神经系统发育,特别是视网膜中müller细胞和脑中星形胶质细胞的发育有促进作用。     

Noninvasive in vivo cell tracking using molecular imaging: A useful tool for developing mesenchymal stem cell-based cancer treatment

Mounting evidence has emphasized the potential of cell therapies in treating various diseases by restoring damaged tissues or replacing defective cells in the body. Cell therapies have become a strong therapeutic modality by applying noninvasive in vivo molecular imaging for examining complex cellular processes, understanding pathophysiological mechanisms of diseases, and evaluating the kinetics/dynamics of cell therapies. In particular, mesenchymal stem cells (MSCs) have shown promise in recent years as drug carriers for cancer treatment. They can also be labeled with different probes and tracked in vivo to assess the in vivo effect of administered cells, and to optimize therapy. The exact role of MSCs in oncologic diseases is not clear as MSCs have been shown to be involved in tumor progression and inhibition, and the exact interactions between MSCs and specific cancer microenvironments are not clear. In this review, a multitude of labeling approaches, imaging modalities, and the merits/demerits of each strategy are outlined. In addition, specific examples of the use of MSCs and in vivo imaging in cancer therapy are provided. Finally, present limitations and future outlooks in terms of the translation of different imaging approaches in clinics are discussed.     

Electron spin resonance and spin trapping technique provide direct evidence that edaravone prevents acute ischemia-reperfusion injury of the liver by limiting free radical-mediated tissue damage

A novel free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), is used for the treatment of acute ischemic stroke and is protective in several animal models of organ injury. We tested whether edaravone is protective against acute liver warm ischemia/reperfusion injury in the rat by acting as a radical scavenger. When edaravone was administered prior to ischemia and at the time of initiation of the reperfusion, liver injury was markedly reduced. Production of oxidants in the liver in this model was assessed in vivo by spin-trapping/electron spin resonance (ESR) spectroscopy. Ischemia/reperfusion caused an increase in free radical adducts rapidly, an effect markedly blocked by edaravone. Furthermore, edaravone treatment blunted ischemia/reperfusion-induced elevation in pro-inflammatory cytokines, infiltration of leukocytes and lipid peroxidation in the liver. These results demonstrate that edaravone is an effective blocker of free radicals in vivo in the liver after ischemia/reperfusion, leading to prevention of organ injury by limiting the deleterious effects of free radicals.     

Hyperbaric Oxygen Therapy Increases Free Radical Levels in the Blood of Humans

It has been postulated that exposure to high concentrations of oxygen results in increased oxygen radical production which may account for the toxic effects of excessive exposure to oxygen. Examination of blood from persons undergoing hyperbaric oxygen (HBO) exposure, by low temperature electron spin resonance (ESR) spectroscopy, demonstrated a marked increase in the magnitude of a signal with properties consistent with a free radical (g = 2.006). The signal diminished to baseline levels within 10 minutes of cessation of HBO exposure. Further in vitro studies of blood revealed an ESR signal generated in red blood cells by oxygen, and dependent on oxyhaemoglobin, which had characteristics indistinguishable from those of the ESR signal of ascorbate radical and the signal in blood from persons undergoing HBO exposure. It is postulated that HBO exposure increases ascorbate radical levels in blood, which is likely to reflect increased ascorbate turnover in human red blood cells.     

Application of in vivo ESR spectroscopy to measurement of cerebrovascular ROS generation in stroke

This study used an in vivo ESR spectroscopy/spin probe technique to measure directly the generation of reactive oxygen species (ROS) in the brain after cerebral ischemia-reperfusion. Transient middle cerebral artery occlusion (MCAO) was induced in rats by inserting a nylon thread into the internal carotid artery for 1 h. The in vivo generation of ROS and its location in the brain were analyzed from the enhanced ESR signal decay data of three intra-arterially injected spin probes with different membrane permeabilities. The ESR signal decay of the probe with intermediate permeability was significantly enhanced 30 min after reperfusion following MCAO, whereas no enhancement was observed with the other probes or in the control group. The enhanced in vivo signal decay was significantly suppressed by superoxide dismutase (SOD). Brain damage was barely discernible until 3 h of reperfusion, and was clearly suppressed with the probe of intermediate permeability. The antioxidant MCI-186 completely suppressed the enhanced in vivo signal decay after transient MCAO. These results clearly demonstrate that ROS are generated at the interface of the cerebrovascular cell membrane when reperfusion follows MCAO in rats, and that the ROS generated during the initial stages of transient MCAO cause brain injury.     

pH control of the chlorophyll a fluorescence in algae

The pH of the suspension medium was found to have a remarkable influence on the “slow” (min) time course of Chlorophyll a fluorescence yield in the green alga Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans. In Chlorella, the decay of fluorescence yield, in the 1- to 5-min region, is strongly retarded at alkaline pH; this decay rate shows an optimum at pH 6–7. In Anacystis, the rise of fluorescence yield, in the same time range, is decreased optimally at pH 6–7; poisoning with 3(3,4-dichlorophenyl)-1,1-dimethylurea reverses the direction of this pH effect. These observations suggest a correlation of the H⁺ status (or the processes associated with it such as photophosphorylation and resulting conformational changes) of the chloroplast to the yield of chlorophyll a fluorescence in vivo.     

In vitro and in vivo studies on oxygen free radical and DNA adduct formation in rat lung and liver during benzo[a]pyrene metabolism

Reactive oxygen species (ROS), possibly produced during the metabolic conversion of benzo(a)pyrene (B[a]P), could be involved in B[a]P-induced genotoxicity and, eventually, carcinogenicity. Therefore, ROS formation by rat lung and liver microsomes was studied in vitro by electron spin resonance (ESR/EPR) spectrometry. B[a]P-mediated generation of ROS was detected in incubations with rat lung, but not with liver microsomes. Inhibition of cytochrome P450 (CYP450) by the non isoform-specific inhibitor SKF-525A resulted in a complete inhibition of B[a]P-dependent ROS formation, whereas ROS formation was not affected by inhibition of prostaglandin H synthase by indomethacin. Subsequently, bulky DNA adduct formation and 8-oxo-dG levels after a single oral dose of B[a]P were examined in vivo in rat lung and liver, in combination with urinary excretion of 8-oxodG. B[a]P exposure resulted in increased urinary 8-oxo-dG levels. On the contrary, 8-oxo-dG levels decreased in liver and lung after B[a]P exposure. Bulky DNA adducts reached higher levels and were more persistent in rat lung than in liver. These results indicate that ROS are generated during the CYP450 dependent metabolism of B[a]P, particularly in the rat lung, but this does not necessarily result in increased levels of oxidative DNA damage in vivo, possibly by induction of DNA repair mechanisms.