Cadmium stimulates mouse skin fibroblast apoptosis by affecting intracellular homeostasis

Context: Cadmium (Cd²⁺) is an important industrial and environmental pollutant and has been shown to induce apoptosis in a variety of cell types and tissues. Objective: To assess the specific effects of low-dose Cd^2+?on the skin. This organ is easily exposed to Cd²⁺, but how it damages cells is not fully understood. Materials and methods: Mouse skin fibroblasts were treated with low doses of Cd^2+?(0.4, 0.8 or 1.6?μM) for 12–48?h, and we observed cell morphological alterations, measured DNA damage and quantified cell viability changes. Results: Cd²⁺-treated fibroblasts exhibited morphological changes and evidence of DNA damage, as well as higher numbers of apoptotic and necrotic cells. There were increased caspase ?3, ?8 and ?9 activities when fibroblasts were treated with 0.4, 0.8 and 1.6?μM CdCl₂ for 24?h. Higher intracellular calcium (Ca²⁺) and reactive oxygen species (ROS) levels, and enhanced efflux of extracellular Ca^2+?and potassium (K⁺). The mitochondrial membrane potential was lowered in treated cells, and the cell cycle arrested in the G0/G1 phase. Bax and Fas gene expression increased and Bcl-2 gene expression decreased. Discussion: The results demonstrate that Cd^2+?exerts typical apoptotic effects in mouse skin fibroblasts. It strongly inhibited proliferation and induced apoptosis in a dose- and duration-dependent manner. Ca^2+?homeostasis was disturbed by oxidative stress, mitochondrial dysfunction and caspase-mediated apoptosis. Conclusion: K^+?efflux and Bax, Bcl-2 and Fas gene expression regulation play important roles in Cd²⁺-induced dysfunction by disrupting intracellular homeostasis in mouse skin fibroblasts.     

Variability of intracellular concentrations of basic fibroblast growth factor in cultured human gliomas.

BACKGROUND: Basic fibroblast growth factor (bFGF) is a small peptide with angiogenic and mitogenic properties that supports the growth and proliteration of human malignant glial tumors in vitro and in vivo in an autocrine fashion. The purpose of this study was to examine the potential relationship between intracellular bFGF concentrations and grade of glial malignancy using a monolayer cell culture system. MATERIALS AND METHODS: Samples from 13 histopathologically verified astrocytic brain tumors and two non-tumorous astrocyte specimens were grown in tissue culture and examined both early and late after explantation together with a bFGF-producing reference cell line. RESULTS: Elevated intracellular concentrations of bFGF were noted in the reference line as well as two of five other glioblastoma multiforme-derived cell cultules, three of five anaplastic glioma-derived cell cultures, and two of three astrocytoma-derived cell cultures. The cells derived from non-tumorous astrocyte specimens expressed low concentrations of bFGF. CONCLUSION: This study demonstrates that overlap exists between the grade ot glial malignancy and intracellular bFGF levels.     

Phosphoramidate peptide inhibitors of human skin fibroblast collagenase

An extensive series of N-(monoethylphosphoryl)peptides was synthesized and their inhibition of purified human skin fibroblast collagenase examined. At the cleavage site S1 all reported compounds have the (EtO)(OK)P(O) group and the peptide side chain extended toward the C-terminal end (up to P5') of the substrate sequence. These phosphoramidates with a tetrahedrally hybridized phosphorus atom are thought to be transition state analogue inhibitors. They exhibited fair inhibitory potency against this vertebrate collagenase having Ki values in the micromolar range. The most potent of these, (EtO)(OK)P(O)-Ile-TrpNHCH3 (68), inhibits with a Ki value of 1.5 microM and is nearly 100 times stronger than (EtO)(OK)P(O)-Ile-Ala-GlyOK (51) (Ki of 140 microM), which has the sequence matching that of the alpha 1 (I) chain of collagen in P1', P2', P3' after the cleavage site. Several compounds were prepared in an attempt to identify the nature of the S2', S3', and S4' binding sites. Alanine at the P2' position was replaced by leucine, phenylalanine, tryptophan, or tyrosine derivatives, resulting in Ki values in a significantly lower range, 1.0-40 microM, compared to 51. No upper size limitation or specificity has been found at this position, yet similar replacements at the P3' position, which is occupied naturally by a glycine residue, gave weaker inhibitors: (EtO)(OK)P(O)-Ile-Tyr(OBzl)-PheOK (57) had a Ki of 120 microM. Hexapeptide derivatives had weaker activities in the 270 microM-2 mM range. All inhibitors were evaluated by using the synthetic thio peptolide spectrophotometric assay.     

人皮肤成纤维细胞系的鉴定及老化检测

目的探索和建立人皮肤成纤维细胞体外分离、培养及鉴定的方法。方法以包皮组织为材料,采用胶原酶消化,分离、培养细胞,倒置显微镜观察,同时进行免疫细胞化学鉴定和β-半乳糖苷酶染色。结果分离的成纤维细胞可在体外快速贴壁生长、增殖。波形蛋白免疫细胞化学染色95%以上为阳性。随成纤维细胞的连续传代培养β-半乳糖苷酶表达呈递增趋势。结论该方法在体外可以获得高纯度的成纤维细胞。     

Practolol inhibits human skin fibroblast cell mat hydroxyproline accumulation

Despite being poorly absorbed practolol (N-4-2-hydroxy-3-(1-methyl-ethyl)-amino propoxy phenyl acetamine) inhibited the accumulation of cell mat hydroxyproline, a measure of collagen synthesis, by human skin fibroblasts (DT2PH) in vitro, (ID50 0.8 X 10(-3) M). The degree of inhibition was dependent on the concentration of practolol used and the incubation time. Neither preinitiation of collagen synthesis nor omitting ascorbic acid from the incubation medium modified this inhibitory action. In contrast, in vitro generated metabolites of practol, using normal and aroclor induced hamster liver preparation, and structural analogues of practolol had no effect on cell mat hydroxyproline levels. Related compounds, propranolol, (1-(isopropylamino)-3(1-naphthyl-oxy)2-propranolol), and paracetamol, (N-(4-hydroxyphenyl)acetamide), both inhibited hydroxyproline levels. Fibroblasts derived from uninvolved skin of a psoriasis patient (PS1) were several fold more sensitive to practolol and propranolol than cells derived from normal skin but showed little change in sensitivity towards paracetamol.     

Xenobiotic metabolism in human skin and 3D human skin reconstructs: a review

In this review, we discuss and compare studies of xenobiotic metabolism in both human skin and 3D human skin reconstructs. In comparison to the liver, the skin is a less studied organ in terms of characterising metabolic capability. While the skin forms the major protective barrier to environmental chemical exposure, it is also a potential target organ for adverse health effects. Occupational, accidental or intended-use exposure to toxic chemicals could result in acute or delayed injury to the skin (e.g. inflammation, allergy, cancer). Skin metabolism may play a role in the manifestation or amelioration of adverse effects via the topical route. Today, we have robust testing strategies to assess the potential for local skin toxicity of chemical exposure. Such methods (e.g. the local lymph node assay for assessing skin sensitisation; skin painting carcinogenicity studies) incorporate skin metabolism implicitly in the in vivo model system used. In light of recent European legislation (i.e. 7(th) Amendment to the Cosmetics Directive and Registration Evaluation and Authorisation of existing Chemicals (REACH)), non-animal approaches will be required to reduce and replace animal experiments for chemical risk assessment. It is expected that new models and approaches will need to account for skin metabolism explicitly, as the mechanisms of adverse effects in the skin are deconvoluted. 3D skin models have been proposed as a tool to use in new in vitro alternative approaches. In order to be able to use 3D skin models in this context, we need to understand their metabolic competency in relation to xenobiotic biotransformation and whether functional activity is representative of that seen in human skin.     

Epoxide metabolism in the liver of mice treated with clofibrate (ethyl-alpha-(p-chlorophenoxyisobutyrate], a peroxisome proliferator

An increase in cytosolic epoxide hydrolase (cEH) activity occurs in the livers of mice treated with peroxisome proliferating-hypolipidemic-nongenotoxic carcinogens. As increases in activity of epoxide metabolizing enzymes may reflect the carcinogenic mechanism, a detailed comparison of the response of cEH, microsomal epoxide hydrolase (mEH), and cytosolic glutathione S-transferase (cGST) activities using the geometrical isomers trans- and cis-stilbene oxide as substrates has been performed in livers from mice treated with clofibrate (ethyl-alpha-(p-chlorophenoxyisobutyrate]. The maximal increase of cEH activity occurred at lower dietary doses of clofibrate (0.5%) and within a shorter time (5 days) than mEH and cGST (2%, 14 days) activity. After 14 days at 0.5% clofibrate, cEH, mEH, and cGST activities were 250, 175, and 165% and 290, 220, and 75% of control values in male and female mice, respectively. Withdrawal of clofibrate from the diet resulted in a reversion of activities to control values within 7 days. Clofibrate treatment shifted the apparent subcellular compartmentation of all three enzymatic activities with an increase in the ratio of soluble to particulate activity. In particular, the relative specific activity of all three enzymes decreased in the light mitochondrial (peroxisomal) cell fraction, and an increase of a mEH-like activity (benzo[a]pyrene-4,5-oxide and cis-stilbene oxide hydrolysis) in the cytosol occurred. Both the increase of cEH activity and the appearance of mEH-like activity in the cytosol are novel responses of epoxide metabolizing enzymes, which may be related to the novel cellular responses that follow clofibrate treatment, peroxisome proliferation, hypolipidemia, and nongenotoxic carcinogenesis.     

The back diffusion of glucose across human skin in vitro

For diabetic patients, blood glucose monitoring is an important part in the management of their disease, however the acquisition of blood requires the use of invasive and often painful methods, and the development of a technique that removes these problems would represent a major advance. The uppermost membrane of the skin, the stratum corneum, has been shown to be the main barrier to percutaneous absorption, but there have been claims that polar water-soluble compounds diffuse across it via aqueous pathways. In this study, skin diffusion cells were used to investigate the back diffusion of tritiated water and the convective transport of 3H-glucose across full thickness human skin after the application of a number of different materials to the stratum corneum. Significant amounts of 3H-glucose back diffused only after complete removal of the stratum corneum by tape stripping, and it is likely that any future attempts to monitor blood glucose levels using non-physical techniques will require a certain degree of damage to the stratum corneum. The extraction through the skin of tritiated water and 3H-glucose after the application of solutions with different osmotic pressures were consistent with the theory that solutions with high osmotic pressures dehydrate the stratum corneum which suggests that passive transport of these radiolabelled molecules through porous pathways was insignificant.     

Tryptophan metabolism in skin diseases.

In a number of skin disturbances conditioned or aggravated by sunlight and/or mainly diffused to the cutaneous connective tissue (present acquired pellagra, lupus erythematosus, porphyria cutanea tarda, actinic reticuloid, Rothmund-Thomson syndrome, lymphocytoma cutis, scleroderma, dermatomyositis, burns, linphomas, parapsoriasis, acrodermatitis enteropathica) excretive changes were found only in the "via kynurenine" metabolites. As a rule kynureniase activity was reduced and tryptophan-pyrrolase activity was increased. In the epidermis tryptophan leads to niacin pathway was found to be present and sometimes autonomous.     

Cerebral glucose metabolism in patients with summer seasonal affective disorder.

Positron emission tomography scans of nine patients diagnosed with summer seasonal affective disorder (SSAD) were compared with scans of 45 normal control subjects to investigate differences in brain glucose metabolism. All subjects performed an auditory discrimination task beginning several minutes before injection of F-18-deoxyglucose and continuing for 30 minutes after injection. Regional glucose metabolic rates were extracted from 60 rectangular regions of interest measured in five planes selected as atlas matches from 28 total slices. Statistically significant differences between patients with SSAD and normal control subjects were found in cerebral glucose metabolic rate and also in normalized regional glucose metabolic rates in the orbital frontal cortex and in the left inferior parietal lobule.     

Purified human plasma glycosaminoglycans limit oxidative injury induced by iron plus ascorbate in skin fibroblast cultures.

A number of studies in vivo and in vitro showed that high levels of glycosaminoglycans (GAGs) are found as a consequence of free radical damage. The GAG over production may represent an endogenous mechanism capable to limit oxidative damage. Based on these hypotheses, the aim of this study was to evaluate the antioxidant property of GAGs of human origin in fibroblast cultures. Purified human plasma GAGs were added to the fibroblast cultures in which oxidative stress was induced by the oxidizing system employing iron (Fe2+) plus ascorbate. We assessed cell death, lactate dehydrogenase activity, membrane lipid peroxidation, DNA damage, protein oxidation, hydroxyl radical (OH*) generation and endogenous antioxidant depletion. The exposure of fibroblasts to FeSO4 produced cell death and increased OH* production. It also caused DNA strand breaks and protein oxidation as shown by the DNA fragment analysis and protein carbonyl content, respectively. In addition, FeSO4 enhanced lactate dehydrogenase activity and lipid peroxidation while decreased antioxidant defences. Purified human GAGs, at three different doses, reduced cell death, limited DNA fragmentation and protein oxidation, decreased OH* generation and lactate dehydrogenase activity, inhibited lipid peroxidation and improved endogenous antioxidant defences. These results further support the hypothesis that these molecules may function as antioxidants.     

纳米二氧化钛对人皮肤基底细胞癌和胚胎皮肤成纤维细胞的生物学效应

目的探讨纳米粒子二氧化钛(TiO2)对体外培养的人皮肤基底细胞癌A431和人胚胎皮肤成纤维细胞的生物学效应。方法TiO2处理体外培养的人皮肤基底细胞癌A431(human skin Basal cell carcinoma,A431)和人胚胎皮肤成纤维细胞(human embryo skin Fibroblast,ESF),MTT法测定细胞生长活性,计数集落形成和荧光染色检测细胞凋亡情况。结果纳米TiO2显著抑制体外培养的人皮肤基底细胞癌A431的集落形成,对其生长活性具有显著负相关性(P<0.05);能够抑制人胚胎皮肤成纤维细胞(ESF)的集落形成,但对其生长活性的抑制无相关性(P>0.05)。结论纳米TiO2可影响体外培养的人皮肤基底细胞癌A431的生长、集落形成,并可导致细胞凋亡。     

Differential regulation on human skin fibroblast by alpha1 adrenergic receptor subtypes

Alpha 1 adrenoceptor (alpha1-AR) regulation of DNA synthesis was studied in human neonatal foreskin fibroblast. Saturation assay with a specific radioligand for alpha1 adrenergic [3H]-prazosin revealed two saturated and specific binding sites with high or low affinity. Competitive binding assay with different antagonist subtypes, defined pharmacologically three major types of alpha1-AR. The alpha1-AR agonists (from 1x10(-10) to 1x10(-4) M) triggered a biphasic action on DNA synthesis reaching maximal stimulation at 1x10(-9) M and maximal inhibition at 1x10(-6) M. Prazosin, abolished the stimulatory (pA2: 9.24) and inhibitory (pA2: 8.80) actions of alpha1-AR agonists. The alpha1-AR stimulation resulted in the activation of phosphoinositide turnover (InsP) via phospholipase C (PLC) involving calcium/calmodulin (CaM) and nitric oxide synthase (NOS) that correlates with the DNA synthesis increment; whereas the inhibition resulted in a decrease of cyclic AMP (cAMP) accumulation via adenylate cyclase inhibition. The potency displayed by the specific antagonists tested in binding, DNA synthesis, InsP and NOS at low agonist concentration suggests that they can be elicited by the activation of the same receptor (alpha1B-AR subtype); while the decrement in DNA synthesis and cAMP at high concentration account by the activation of alpha1D-AR coupled to Gi protein. Non-functional alpha1A-AR in neonatal human foreskin fibroblast was observed. Results suggest that the expression of alpha1-AR subtypes on human skin fibroblast may differentially activate signaling pathways that modulate physiological response of the cells.     

PDGF-BB和CTGF在体外对人皮肤成纤维细胞增殖的影响

目的探讨重组人血小板衍生生长因子（rhPDGF-BB）和重组人结缔组织生长因子（rhCTGF）单独或联合应用对体外培养的人皮肤成纤维细胞（human skin fibroblast,HSF）增殖的影响,为寻求快速扩增HSF的方法提供参考依据。方法取手术中切除的正常皮肤,以常规组织块法体外获得皮肤成纤维细胞。取生长良好的第4~7代细胞种入96孔板,分实验组和对照组,分别加入不同浓度的rhPDGF-BB、rhCTGF或rhPDGF-BB＋rhCTGF作用。于第1、3、6、9d采用四唑盐（MTT）法分别测定各组吸光度A值,观察上述两种生长因子对细胞的增殖作用。结果 rhPDGF-BB能明显促进体外培养的HSF增殖,在1~9d,0.1ng/ml~100ng/ml范围内呈浓度时间依赖关系。rhCTGF也能明显促进HSF增殖,除第1d最大效应浓度为50ng/ml,其余时间点在10ng/ml~100ng/ml范围内呈浓度时间依赖关系。而rhCTGF150ng/ml组的增殖效果相对100ng/ml组略成下降趋势。rhPDGF-BB与rhCTGF最大与最小效应联用组亦成浓度时间依赖关系。最大效应联用组的增殖效果与以上两种生长因子单独应用比较均具有统计学意义（P〈0.05）。最小效应联用组与rhCTGF10ng/ml组比较有统计学意义（P〈0.05）,而与rhPDGF-BB0.1ng/ml组比较无统计学意义（P〉0.05）。另外,最大效应浓度的两生长因子单独应用时,rhPDGF-BB促增殖效果优于rhCTGF（P〈0.05）。结论 rh-PDGF-BB和rhCTGF均可促进体外培养的HSF增殖,在一定范围内呈浓度时间依赖关系,而且前者优于后者。两者联合应用对HSF增殖具有协同效应。     

Permeation, metabolism and site of action concentration of nicotinic acid derivatives in human skin. Correlation with topical pharmacological effect.

A novel methodology for establishing a pharmacological dose-effect relationship of methyl nicotinate, hexyl nicotinate and nicotinic acid acting as peripheral vasodilators in the skin following topical application is investigated. This methodology involves the estimation of the unbound drug concentration in the aqueous compartment at the site of action in tissue, termed C(*), which was evaluated as the pertinent concentration responsible for the pharmacological effect. Blood capillaries next to the epidermis-dermis boundary were postulated to be the relevant site of action. C(*) was estimated from drug transport parameters for different layers of human cadaver skin determined in vitro. Immunohistochemical studies showed that the plane of separation of skin achieved by heat treatment was between the basal cells of the epidermis and the lamina lucida, confirming the integrity of the epidermis and the dermis used in the experiments. The permeation rate for epidermis increased drastically with increasing lipophilicity of the drug. Dermis permeability was roughly the same for all three compounds. The epidermis represented the major transport barrier in vitro for methyl nicotinate and nicotinic acid but not for hexyl nicotinate. The esters were metabolised to nicotinic acid during tissue permeation to an extent that was rather limited for the epidermis but very pronounced for the dermis. Nonspecific alpha-naphthylacetate-esterase activity was predominantly located in the dermis, which was in agreement with the metabolism results. The drugs were applied each at three different concentrations in vivo to the ventral forearm of healthy human volunteers and vasodilation was evaluated based on skin erythema which was quantified by measuring colour change of reflected light. Area under the curve of the change of colour co-ordinates as a function of time was used as a measure of pharmacological effect. The pharmacological effect of all three drugs was comparable when similar C(*) values were considered, even though the concentrations applied to the skin differed by orders of magnitude. The effect showed a strong positive dependence on C(*). Methyl and hexyl nicotinate showed identical, nearly sigmoidal effect/C(*)-profiles, while the profile for nicotinic acid was linear, suggesting a possible difference in the intrinsic pharmacological potency between the esters and the acid. These results demonstrate the validity of C(*) as the relevant drug concentration for the cutaneous pharmacological effect of the topically applied drugs and underline the usefulness of the presented methodology for establishing dose-response relationships in dermal therapy and expressing bioavailability.     

Lead induces Siberian tiger fibroblast apoptosis by interfering with intracellular homeostasis

Lead (Pb²⁺) is a poisonous heavy metal that causes many pathophysiological effects in living systems. Its toxicological effects are well known as it causes apoptosis of several cell types and tissues. This study aimed to determine the criteria required for early diagnosis of Pb²⁺ poisoning in the Siberian tiger using a tiger population in China, to identify a safety Pb²⁺ concentration threshold, and to provide suggestions for preventing Pb²⁺ poisoning in Siberian tigers. We investigated the apoptotic effects of Pb²⁺ (0, 32, 64, and 125?μM) for 12–48?h on Siberian tiger fibroblasts in vitro. Typical apoptotic effects were observed after Pb²⁺ exposure. Pb²⁺ strongly blocked DNA synthesis in the G0/G1 phase and induced cell apoptosis in a dose- and time-dependent manner. Intracellular free calcium (Ca²⁺) levels, reactive oxygen species levels, and efflux of extracellular Ca²⁺ were increased. The mitochondrial membrane potential was lowered. Caspase-3, -8, and -9 activities were increased when fibroblasts were treated with 32, 64, and 125?μM Pb²⁺. The gene expression levels of Bax, caspase-3, -8, Fas, and p53 were increased, while that of Bcl-2 was decreased. Calcium homeostasis and mitochondrial function were disturbed. Ca²⁺ efflux, oxidative damage, activation of caspases, and regulation of Bax, Bcl-2, caspase-3, -8, Fas, and p53 gene expression played an important role in the apoptotic effects. The disorder of intracellular homeostasis was the trigger for apoptosis in Siberian tiger fibroblasts.     

Mechanisms of metformin action on glucose transport and metabolism in human adipocytes

The mechanisms of metformin effects on glucose transport and metabolism were investigated in human adipocytes. Human preadipocytes obtained from surgical biopsies were differentiated in vitro into adipocytes and the effects of metformin on glucose uptake, glucose oxidation and the involved signaling pathways were analyzed. Metformin (1 mM, 24 h) increased glucose uptake 2.3 ± 0.2-fold (p < 0.001 vs. basal) in human adipocytes, without altering cell viability and oxygen consumption. Metformin did not alter GLUT-1 mRNA expression and protein content but increased GLUT-4 mRNA expression and cellular protein content, leading to increased GLUT-4 protein content in the plasma membrane. Neither basal nor insulin-induced phosphorylation of Akt at Ser-473 and AS160 (Akt substrate of 160 kDa) at Thr-642 were enhanced by metformin. Suppression of metformin-induced AMP-activated protein kinase (AMPK) activity by AMPKα1 silencing, however, reduced metformin-associated GLUT-4 expression and stimulation of glucose uptake. In addition, metformin induced glucose oxidation. In conclusion, activation of AMPKα1 without impairment of cell respiration is crucial for metformin-mediated increase in GLUT-4 protein content and glucose uptake in human adipocytes.     

Modification of human airway smooth muscle reactivity by drugs that interfere with arachidonic acid metabolism

Histamine-induced contractions of small airways from human lung were substantially augmented by the cyclo-oxygenase inhibitor, indomethacin, whereas the contraction of larger airways was not. Mixed cyclo-oxygenase/lipoxygenase inhibitors of arachidonic acid metabolism did not modify histamine-induced contractions of smooth muscle from either small or large airways but completely reversed the augmentation of histamine responses produced by indomethacin on the smaller airways. The SRS-A (slow reacting substance of anaphylaxis) antagonist, FPL55712, did not affect the augmentation of histamine-induced contractions by indomethacin.     

Glucose and lipid metabolism during long-term treatment with cilazapril in hypertensive patients with or without impaired glucose metabolism.

The effects of long-term cilazapril therapy on glucose tolerance and serum lipid profiles were investigated in 19 hypertensive patients: seven with normal glucose tolerance and 12 with glucose intolerance (including three patients with noninsulin-dependent diabetes). Cilazapril was administered once daily for a mean duration of 6.4 months. A 75-g oral glucose tolerance test was performed before and during long-term therapy with cilazapril. Cilazapril produced satisfactory control of blood pressure in both patient groups during long-term therapy. It was well tolerated by all patients. Neither fasting nor postglucose-load venous plasma glucose levels were altered in either group of patients, and no patients with normal glucose tolerance developed diabetes mellitus during the study. There were no significant changes in the insulinogenic index (delta IRI/delta BS at 30 min postglucoseload) in patients with normal or impaired glucose tolerance. No significant changes in fasting levels of serum cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides were observed in either group. These results suggest that effective long-term cilazapril therapy does not compromise glucose or lipid metabolism in hypertensive patients. Cilazapril may have a clinical advantage in that it can be given to hypertensive patients without concern that it might alter their serum lipid concentrations or impair glucose tolerance.     

Inflammation of the skin. I. Phospholipid metabolism in some experimental inflammations of mouse skin.

Phospholipid metabolism in inflamed tissue of the mouse skin which had been induced by the application of 1-chloro-2, 4-dinitrobenzene (DNCB), croton oil, or irradiation of ultraviolet rays was examined, and it was found that phospholipid levels had increased in theinflamed tissues. In the case of ultraviolet rays, the increase was temporary, and the level returned to that of control after 3 or 4 days. In the case of DNCB or croton oil, the level increased after a decrease for a short period. The pattern of the increase between physical and chemical irritation was different. Increase of incorporation of 32-P into phospholipid in inflamed tissue was examined, and it was observed that the level reached a maximum after one day. It is thus assumed that phospholipid plays an important role in the mechanism of inflammation.