Inhibitory effect of Hibiscus protocatechuic acid on tumor promotion in mouse skin.

Hibiscus protocatechuic acid (PCA), a phenolic acid isolated from Hibiscus sabdariffa L., was evaluated for its ability to inhibit the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion in skin tumors of female CD-1 mice. Topical application of PCA (5, 10 or 20 micromol) 5 min prior to TPA (15 nmol) treatment twice weekly for 20 weeks to mice which were initiated with benzo[a]pyrene (B[a]P) inhibited the incidence of tumors in mice to 81.3, 62.5 and 56.3%, respectively, while all mice in the TPA-treated group developed tumors. The average number of tumors in mice pretreated with PCA was 2-4 and that of mice treated only with TPA was 6.6. The protection effects of PCA were also presented by its significant suppression on the TPA-induced hyperplasia in the skin and edema of mouse ears by 65 and 73% at doses of 10 and 20 micromol, respectively. When it was applied to the dorsal surface of CD-1 mice before TPA application, PCA (5, 10 or 20 micromol) inhibited the induction of epidermal ornithine decarboxylase (ODC) activity by 5 nmol TPA and myeloperoxidase (MPO) activity by 6.5 nmol TPA. The same doses of PCA also reduced the formation of hydrogen peroxide in the mouse skin to an inhibition of 61, 84 and 89%, respectively, when compared with that of the TPA-treated group. These results indicate that PCA possesses potential as a cancer chemopreventive agent against tumor promotion.

Cytochromes P-45011 beta, P-450scc and adrenodoxin gene expression during adrenocortical regeneration in the rat.

Circulating levels of 11-deoxycorticosterone are increased during the development of adrenal-regeneration hypertension. The present studies were undertaken to examine the mechanisms involved in this increase. Plasmids containing cDNA inserts coding for cytochrome P-45011 beta, cytochrome P-450scc and adrenodoxin were used to determine, by Northern blot analysis, mRNA levels at 1, 2 and 3 weeks of adrenal regeneration. There was a striking decrease in mRNA transcripts for all three enzymes during the first week of regeneration when compared with intact adrenal tissue. Over the next 2 weeks the mRNA levels increased to 64% for P-45011 beta, to 80% for P-450scc and to 82% for adrenodoxin. Actin mRNA levels were 70% of control levels in the first week but were back to control levels by the second week. The present findings suggest that decreased expression of steroid 11 beta-hydroxylase could contribute to the increased secretion of 11-deoxycorticosterone during the early stages of adrenal regeneration.

Specific induction of glutathione S-transferase GSTM2 subunit expression by epigallocatechin gallate in rat liver.

The antitumor effect of green tea polyphenols has been well characterized in numerous papers. However, the mechanism of their action is still poorly defined. In this study, epigallocatechin gallate (EGCG), the main ingredient of green tea extract, was studied for its effect on the expression of glutathione S-transferases (GSTs) in rat liver to examine the mechanism of action. Liver samples were collected from Sprague-Dawley rats treated with EGCG in H(2)O by portal vein perfusion and examined for total GST activity and GST expression. The results showed that the induction of GST activity by EGCG was dose- and time-dependent. GST activity was increased about 28-fold at 12 hr after treatment. Three GST subunits (GSTA1/2, GSTM1, and GSTM2) were examined by Western blot for changes in protein level affected by EGCG (1 mg/kg weight). Only GSTM2 revealed a significant time-dependent increase, with a maximal induction of approximately 2.0-fold. The differential effect of EGCG on GST subunit expression was also verified by immunocytochemical examination and showed strong induction of the GSTM2 (but not the GSTA1/2 and GSTM1) level in liver section. This induction occurred as early as 3 hr after treatment and extended gradually outward from the hepatic veins as treatment time increased. The change in the GSTM2 protein level was accompanied by a corresponding alteration in mRNA quantity ( approximately 2.0-fold of control). Our report is the first to demonstrate a specific induction of the GSTM2 subunit by a chemopreventor and suggests a primary influence of EGCG on GSTM2 gene expression.

Induction of apoptosis by hibiscus protocatechuic acid in human leukemia cells via reduction of retinoblastoma (RB) phosphorylation and Bcl-2 expression.

Hibiscus protocatechuic acid (PCA), a phenolic compound isolated from the dried flower of Hibiscus sabdariffa L. (Malvaceae), demonstrated antioxidant and antitumor promotion effects in our previous study. In the present study, Hibiscus PCA was found to inhibit the survival of human promyelocytic leukemia HL-60 cells in a concentration- and time-dependent manner. The study revealed that HL-60 cells underwent internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after a 9-hr treatment with Hibiscus PCA (2 mM). Flow cytometric analysis of the DNA content of cells treated with PCA for 12 hr showed that the cells were distributed mainly in the hypodiploid phase (apoptotic peak, 46.7%), less in the G(1) (34.2%) and S phase (14.0%), and few in the G(2)/M phase (5.1%). Moreover, PCA treatment caused an increase in the level of hypophosphorylated retinoblastoma (RB; 180% of control at the 6-hr time point) and, on the contrary, a decline in hyperphosphorylated RB. A rapid loss of RB was observed when the treatment period was extended. Further studies showed that Hibiscus PCA application reduced Bcl-2 protein expression to 47%, and increased Bax protein expression to 181% after 1.5 hr as compared with time 0. Overexpression of Bcl-2 in HL-60 cells delayed the occurrence of Hibiscus PCA-induced apoptosis. These data suggest that Hibiscus PCA is an apoptosis inducer in human leukemia cells, and that RB phosphorylation and Bcl-2 protein may play a crucial role in the early stage.

Evidence of multi-step regulation of HSP72 expression in experimental sepsis.

Heat shock proteins (Hsps) are a family of highly conserved proteins induced in response to various stresses. Hsps protect cells against subsequent lethal circumstances. Previous work from our laboratory has indicated that Hsp72 is not induced during experimental sepsis in rats, but the regulation of the induction of Hsp72 synthesis in this disease cascade has not been investigated. In the present study, we evaluated the expression of the hsp72gene, focusing on the activation and DNA-binding ability of heat shock factor 1 (HSF1), hsp mRNA accumulation, and Hsp72 synthesis in animal sepsis models induced by cecal ligation and puncture procedure. The results were compared with those of sham-treated and heat-shocked rats. It was shown that the expression of the hsp72 gene in sepsis was a multi-step process, as previously documented in in vitro studies. Hsp72 synthesis was not induced during sepsis, whereas DNA binding of HSF was detectable, suggesting that the induction of Hsp72 is blocked downstream to HSF-DNA complex formation by the metabolic alteration occurring during sepsis. The dissociation failure of the constitutive heat shock element binding factor (CHBF) from the heat shock element may play an important role in this negative regulation.

Significant differences in serum activities of matrix metalloproteinase-2 and -9 between HCV- and HBV-infected patients and carriers.

To examine the possible involvement of MMP-9 and -2 in the development of liver diseases caused by HCV or HBV infection, serum activities of both enzymes were studied by zymograph. Eight groups of subjects (60 for each) were examined in the study: healthy control, patients with hepatoma, liver cirrhosis, chronic hepatitis B or chronic hepatitis C, and carriers positive for HBsAg, both HBsAg and HBeAg, or anti-HCV. The results showed significant changes in the MMP-9 and -2 activities in the carriers. The presence of HBeAg was accompanied by a highest activity of MMP-2 and an inversely correlated (r=-0.578, P=<0.001), lowest activity of MMP-9 among all groups. For those with active liver diseases, MMPs activities were fluctuated at each stage of pathological symptoms. Chronic hepatitis B and C patients had significant different serum MMP-2 and -9 activities. These findings imply an influence on the balance of MMPs system by the existence of virus that might influence the following progression of liver disease, and a distinction between the pathological mechanisms of HCV and HBV. Since the serum MMPs activities were significantly varied between each stage of liver disease, an individual profile of these parameters might serve as an easy accessing serum marker to monitor the progression of liver disease.

Fluctuation of serum leptin level in rats after ovariectomy and the influence of estrogen supplement.

In order to understand the mechanism of increasing body fat in perimenopausal and postmenopausal women, an ovariectomy-induced obesity model was used to study the role of leptin. In this investigation, a long-term study lasted for 13 weeks was conducted to monitoring the change of serum leptin level in rats after the loss of estrogen, and also to examine the influence of estrogen replacement. The results showed that three weeks after the removal of ovaries the body weight of Ovx rats was already significantly higher than the other two groups, and continued to gain more weight thereafter. Accompanying with the significant weight gain was the changes in the serum leptin levels. The leptin concentration declined gradually during the first half of experimental period, dropping down to an almost undetectable level at week 7 (0.216+/-0.132 ng/ml). Subsequently, its concentration began to elevate, and by the end of experiment leptin level was significantly higher (3.182+/-0.936 ng/ml) than the value before the operation (0.818+/-0.242 ng/ml). This fluctuation of serum leptin level caused by ovariectomy was eliminated by the replacement of estrogen. The present data indicate that ovariectomy-induced weight gain is caused by the early drop in leptin level. The later rise in leptin production is connected to the increased body weight probably originated from a reduced sensitivity in leptin signal.

Suppression of the TPA-induced expression of nuclear-protooncogenes in mouse epidermis by crocetin via antioxidant activity.

Crocetin, a major component of the fruit of Gardenia jasminoides Ellis, was investigated for its antitumor promoting effect on 12-O-tetradecanoylphorbol-13-acetate-promoted mouse skin carcinogenesis. Topical application of 5 nmol TPA to CD-1 mice once daily for 5 days caused epidermal hyperplasia, and increases in the levels of c-Fos, c-Jun and c-Myc in the suprabasal layer of epidermis and the muscle layer of dermis. Immunocytolochemical examination showed that pretreatment of 1 mumol crocetin repressed the TPA-induced epidermal hyperplasia and the expressions of c-Jun, c-Fos and c-Myc to the extent of 47, 44 and 45% respectively. Crocetin of 3.0 mumol exhibited stronger inhibition on the induced hyperplasia and the oncoproteins levels (by 60, 53 and 55% respectively). Western blotting analysis confirmed this inhibitory effect of crocetin. Pretreatment of crocetin also repressed the TPA-induced H2O2 production and myeloperoxidase activity. These data indicate that crocetin suppresses the TPA-induced skin carcinogenesis maybe via its antioxidant property which, in turn, leads to a reduction in the TPA-induced expressions of c-Jun, c-Fos and c-Myc in mouse epidermis.

Potential effect of sodium nitrite on the expression of nuclear proto-oncogenes during 2-acetyl aminofluorene-induced hepatocarcinogenesis in rats.

2-acetyl aminofluorene (AAF) reacts in acidic conditions with nitrous fume yielding N-nitroso-AAF (N-NO-AAF), as previously described, that exerts more toxic and mutagenic effects than its parental compound. In this study, the effect of sodium nitrite (NaNO2) on the tumorigenicity of AAF in rats fed with AAF and NaNO2 was observed. Wistar rats were divided into five groups: group I served as control; group II were treated with NaNO2 (0.3%); group III was given 0.02% AAF alone; groups IV and V received both AAF and NaNO2 (0.2 and 0.3% respectively) in their diet for 12 weeks. At the end of the experiment, all rats in groups III, IV and V developed early stage phenomena of hepatocellular carcinoma, including hepatomegaly with variable-sized foci and neoplastic nodules. Severe damage was observed in the rats treated with AAF and NaNO2. Feeding of AAF (0.02%) for 3 months elevated the levels of c-Fos, c-Jun and c-Myc proteins in the rat livers. The AAF-induced c-Jun, c-Fos and c-Myc expressions were significantly magnified (P < 0.001) by NaNO2. These data confirmed that the strengthening of AAF-induced hepatocarcinogenesis by NaNO2 should be associated with its enhancing effect on the AAF-induced increases in the expressions of c-Jun, c-Fos and c-Myc.

Suppressive effect of penta-acetyl geniposide on the development of gamma-glutamyl transpeptidase foci-induced by aflatoxin B(1) in rats.

The suppressive effects of penta-acetyl geniposide, (Ac)(5)-GP, on the hepatotoxic lesions-induced by aflatoxin B(1) (AFB(1)) were investigated in male Wistar rats. Rats were divided into six groups: groups I and II served as normal and solvent control, respectively; group III was given AFB(1) (2 mg/kg body weight) alone; group IV was given (Ac)(5)-GP (2 mg/kg) alone; and groups V and VI received both AFB(1) (2 mg/kg body weight) and (Ac)(5)-GP (1 mg and 2 mg/kg body weight, respectively). Rats received treatments for 8 weeks, then were maintained on basal diet for 32 weeks. At the end of the experiment (week 40), the liver lesions (e.g. fatty change, eosinophilic and bile duct dilation) and preneoplastic changes in rats of groups V and VI were reduced when they were compared with group III. There were no liver lesions and preneoplastic changes in rats treated with (Ac)(5)-GP alone. Although no differences in the total number of gamma-glutamyl transpeptidase (GGT)-positive foci was observed between the groups treated with AFB(1) along with or without (Ac)(5)-GP, the treatment of (Ac)(5)-GP significantly reduced the number of AFB(1)-induced GGT positive foci (with diameter larger than 0.3 mm). These results indicated that the protective effect of (Ac)(5)-GP on early hepatocarcinogenesis-induced by AFB(1) was associated with the inhibition of GGT foci development.

Protective effects of dried flower extracts of Hibiscus sabdariffa L. against oxidative stress in rat primary hepatocytes.

Dried flower extracts of Hibiscus sabdariffa L., a local soft drink material and medical herb, was found to possess antioxidant activity in the present study. In the preliminary studies, antioxidant potential of three fractions of the ethanol crude extract (HS-C: chloroform-soluble fraction; HS-E: ethyl acetate soluble fraction; HS-R: residual fraction) obtained from the dried flowers of Hibiscus sabdariffa L. were evaluated by their capacity of quenching 1,1 -diphenyl-2-picrylhydrazyl (DPPH) free radical and inhibiting xanthine oxidase (XO) activity. HS-E showed the greatest capacity of scavenging free radical (EC50=0.017mg/ml), and HS-C showed the strongest inhibitory effect on XO activity (EC5o=0.742 mg/ml). Furthermore, antioxidant bioactivities of these crude extracts were investigated using a model of tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat primary hepatocytes. All fractions were found to inhibit significantly the unscheduled DNA synthesis (UDS) induced by t-BHP at a concentration of 0.20 mg/ml. HS-C and HS-E also decreased the leakage of lactate dehydrogenase (LDH) and the formation of malondialdehyde (MDA) induced by t-BHP (1.5 mM) considerably at a concentration of 0.10 and 0.20 mg/ml in the rat primary hepatocyte cultures. These results indicated that the dried flower extracts (HS-C and HS-E) of H. sabdariffa L. protect rat hepatocytes from t-BHP-induced cytotoxicity and genotoxicity by different mechanisms.

Protective effect of Hibiscus anthocyanins against tert-butyl hydroperoxide-induced hepatic toxicity in rats.

Hibiscus anthocyanins (HAs), a group of natural pigments occurring in the dried flowers of Hibiscus sabdariffa L., which is a local soft drink material and medical herb, were studied for antioxidant bioactivity. The preliminary study showed that HAs were able to quench the free radicals of 1,1-diphenyl-2-picrylhydrazyl. This antioxidant bioactivitiy was further evaluated using the model of tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity in rat primary hepatocytes and hepatotoxicity in rats. The results demonstrated that HAs, at the concentrations of 0.10 and 0.20 mg/ml, significantly decreased the leakage of lactate dehydrogenase and the formation of malondialdehyde induced by a 30-min treatment of t-BHP (1.5 mM). The in vivo investigation showed that the oral pretreatment of HAs (100 and 200 mg/kg) for 5 days before a single dose of t-BHP (0.2 mmol/kg, ip) significantly lowered the serum levels of hepatic enzyme markers (alanine and aspartate aminotransferase) and reduced oxidative liver damage. The histopathological evaluation of the liver revealed that Hibiscus pigments reduced the incidence of liver lesions including inflammatory, leucocyte infiltration, and necrosis induced by t-BHP in rats. Based on the results described above, we speculate that Hibiscus pigments may play a role in the prevention of oxidative damage in living systems.

Heat shock treatment decreases the mortality of sepsis in rats.

Sepsis is an increasingly common and lethal diagnosis in hospitalized patients. In spite of the advances in antibiotics and medical equipment, the mortality rate has not been improved in the last decade. Recently, heat shock proteins (Hsps) have been well documented to play a self-protective role in almost all living cells under various pathological and physiological stresses through a mechanism known as thermotolerance or cross tolerance. The present study was designed to investigate the expression of Hsp72 and the protective role of pre-induction of Hsps in the mortality during different phases of sepsis. Adult male Sprague-Dawley rats were employed in the study. Sepsis was induced by cecal ligation and puncture (CLP). Heat shock treatment was performed 16 hrs before sepsis induction by heating the rats whole-bodily with an electric heating pad under general anesthesia. The mortality rates with time in both control and preheated groups were monitored. Hsp72 was detected by SDS-PAGE, Western blotting and immunostaining in the brain, heart, liver, kidney, lung, adrenal gland, muscle and lymphocytes. The results show that both early (9 hrs post-CLP) and late (18 hrs post-CLP) sepsis failed to increase the synthesis of Hsp72. Previous induction of Hsps by heat shock treatment significantly decreased the mortality rate of late sepsis. Applying a sufficient inducer to lymphocytes of late sepsis reversed the synthesis of Hsp72 from inactive state into an over-expressive situation in vitro. These results suggest that Hsps are involved in the pathogenesis of sepsis and the involvement of Hsps during the progression of sepsis could add to a first line of host defense against invasive pathogens. Searching for an acceptable agent or less invasive method that leads to increased Hsps expression may provide a means for better treatment of severe infection.

The gene expression of cytochrome P-450(11) beta during adrenal regeneration in the rat: studies using in situ hybridization.

Low levels of mRNA for the key adrenal steroidogenic enzyme, cytochrome P-450(11) beta, have been reported in the early stages of adrenal regeneration. To determine whether the defect in cytochrome P-450(11) beta gene expression is located in the adrenal zona fasciculata or glomerulosa, in situ hybridization techniques have been employed. Sections from 1 week regenerating adrenals were probed with sense and anti-sense cytochrome P-450(11) beta RNA. The signal was intense in the zona fasciculata cells in control adrenals but very low in those of adrenal regenerates. These data establish that the low levels of mRNA transcripts of cytochrome P-450(11) beta are localized to the zona fasciculata cells responsible for 11-deoxycorticosterone secretion in regenerating adrenals.

Inhibition of protein kinase C and proto-oncogene expression by crocetin in NIH/3T3 cells.

Crocetin, a carotenoid isolated from the seeds of Gardenia jasminoides, was found to be a potent inhibitor of tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin. When mouse fibroblast NIH/3T3 cells were treated with TPA alone, protein kinase C (PKC) translocated from the cytosolic fraction to the particulate fraction. Pretreatment with 60 and 120 microM crocetin for 15 min inhibited the TPA-induced PKC activity in the particulate fraction by 50% and 66%, respectively, but did not affect the level of PKC protein. Crocetin also reduced the level of TPA-stimulated phosphorylation of cellular proteins. Cells pretreated with crocetin (120 microM) had 55% less PKC [3H]phorbol dibutyrate-binding capacity. Suppression of TPA (100 ng/mL)-induced c-jun and c-fos gene expression was also observed in the mouse fibroblast cells pretreated with crocetin (30, 60, and 120 microM). Our results provided a basis for understanding the inhibitory effect of crocetin on TPA-mediated tumor promotion.

Inhibitory effect of atractylon on tert-butyl hydroperoxide induced DNA damage and hepatic toxicity in rat hepatocytes.

Atractylon, a main sesquiterpenic constituent of Atractylodes rhizomes, was studied for the mechanism of its inhibitory effects on the tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity and lipid peroxidation in primary culture of rat hepatocytes. In the preliminary study, atractylon showed an effective antioxidant property tested by its capacity for quenching 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Further investigations showed that atractylon at the concentrations of 0.01, 0.1 and 1.0 mg/ml decreased the formation of malondialdehyde (MDA), leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) and repair synthesis of DNA induced by 30-min treatment of t-BHP (1.5 mM) in primary cultured rat hepatocytes. Addition of atractylon also attenuated the genotoxicity of t-BHP evaluated by unscheduled DNA synthesis. The sum of the results suggested that the protective effect of atractylon against oxidative stress induced by t-BHP is via its ability to quench free radicals.

Proteolytic activities of mouse sarcoma 180 cells that are inhibited by Bowman-Birk and Kunitz protease inhibitors.

In this study, using zymogram analysis two proteolytic activities were identified in the mouse sarcoma 180 (S-180) cells that were activated by trypsin treatment and inhibited by both BBI and ACTI. These enzymes, with molecular weights of 46 kDa (dominant band) and 62 kDa (minor band), were mainly localized in the cytosol, and had optimal activity at pH 7 and 8 respectively. Their inhibition by DFP, BBI and ACTI but not EDTA and TPCK indicated they were trypsin-like serine proteases and may be the intracellular target-enzymes of protease inhibitors. The level of the precursor of the 62 kDa protease was significantly increased in the S-180 solid and soft tumors, whereas the level of the 46 kDa precursor was almost undetectable, implying that a physiological role may be played by these serine proteases during tumor invasion.

Hibiscus protocatechuic acid inhibits lipopolysaccharide-induced rat hepatic damage.

Hibiscus protocatechuic acid (PCA), a phenolic compound found in the dried flowers of Hibiscus sabdariffa L. (Malvaceae), was demonstrated to have an antioxidant effect in vitro and in vivo, and an antitumor property in our previous study. In the present study, we used lipopolysaccharide (LPS, an endotoxin) to induce rat liver inducible nitric oxide synthase (iNOS), and found that pretreatment with PCA decreased the liver iNOS and the serum total nitrite induced by LPS. Our investigation showed that pretreatment of rats with PCA (0.2 and 0.5 mmol/kg dosed by gavage) for 5 days significantly decreased the serum levels of the hepatic enzyme markers alanine- and aspartate aminotransferase (ALT, alanine aminotransferase; AST, aspartate aminotransferase) induced by the 6-h treatment with LPS (i.p.; 5 mg/kg). Histopathological evaluation of the rat livers revealed that PCA reduced the incidence of liver lesions induced by LPS, including neutrophil infiltration, congestion, and liver cell swelling induced by LPS in rats. We conclude that PCA, an antioxidant, presents an inhibitory potential on iNOS and hepatic damage induced by LPS.

Protective effects of capillarisin on tert-butylhydroperoxide-induced oxidative damage in rat primary hepatocytes.

Capillarisin (Cap), a main constituent of Artemisia capillaris (Compositae), was studied for its antioxidant bioactivity. In the preliminary study, Cap expressed a antioxidant property by its capacity for quenching the free radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH). This antioxidant bioactivity of Cap was investigated further using a model of t-butylhydroperoxide (t-BHP)-induced cytotoxicity and genotoxicity in rat primary hepatocytes. Results presented here demonstrate that Cap, at concentrations of 0.01-1.00 mg/ml, significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) and the formation of malondialdehyde (MDA) induced by 30 min treatment of t-BHP (1.5 mM) in primary cultured rat hepatocytes. Cap also attenuated the t-BHP-induced diminution of glutathione (GSH) and high level of DNA repaired synthesis. These results lead to speculation that Cap presents inhibitory effects against t-BHP-caused cytotoxicity and genotoxicity in rat primary hepatocyte cultures at least via two distinct pathways, stabilizing the GSH system and quenching free radicals.

L ferritin accumulation in macrophages infiltrating the lung during rat Angiostrongylus cantonensis infection.

A 22-kDa protein was increased quantitatively, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques, in lung microsomes prepared from Angiostrongylus cantonensis-infected rats. However, it was almost absent in normal rats. The protein was purified by sequential chromatography on Superdex 200 columns and identified chemically and immunologically as ferritin. Using isoelectric focusing and anion exchange chromatography, it was identified as L ferritin. Distribution of this 22-kDa protein in the lung tissue of A. cantonensis-infected rate was studied by immunocytochemistry. Positively stained cells were mainly infiltrated macrophages. Our results suggest that L ferritin accumulation in the macrophages may be related to the proliferation of connective tissue elements and the inflammatory response to A. cantonensis dwelling in the pulmonary arteries of the rat.