Effects of a Phytogenic Feed Additive Versus an Antibiotic Feed Additive on Oxidative Stress in Broiler Chicks and a Possible Mechanism Determined by Electron Spin Resonance.

Phytogenic feed additives are plant-derived products used in poultry feeding to improve overall performance of broilers. In this study, 588 one day-old Cobb 500 chicks were fed one of four diets and housed on either dirty or clean litter for 3wks. Treatments included: Group I: commercial diet with no additive and housed on clean litter; Group II: commercial diet with no additive and housed on dirty litter; Group III: commercial diet with a 0.05% inclusion of the anitobiotic, BMD (bacitracin methylene disalicylate); Group IV: commercial diet with a 0.05% inclusion of a phytogenic feed additive (PFA). The study was designed around a random block assignment of treatments allocated to groups of twenty-one birds per pen. Blood samples were obtained from chicks at 18 days of age for measurement of leukocyte oxidative activity by a bioluminescence technique. Results of the study showed that chicks in the treatment groups fed the PFA had significantly lower oxidative stress (p<0.02) when compared to the BMD treatment group. Once this was determined, electron spin resonance (ESR) spin trapping was used to detect and measure hydroxyl or superoxide radicals in. Fenton chemistry was utilized for production of hydroxyl radicals and a xanthine/xanthine oxidase reaction for the production of superoxide radicals in the diet and in RAW 264.7 mouse peritoneal monocytes exposed to the diet. Results from the reactions showed that the antibiotic scavenges hydroxyl and superoxide radicals more efficiently than the phytogenic. The results were comparable to those measured in the RAW 264.7 cells.

Isoflurane impairs odour discrimination learning in rats: differential effects on short- and long-term memory.

BACKGROUND: Anaesthetics suppress the formation of lasting memories at concentrations that do not suppress perception, but it is unclear which elements of the complex cascade leading from a conscious experience to a lasting memory trace are disrupted. Experiments in conscious humans suggest that subhypnotic concentrations of anaesthetics impair consolidation or maintenance rather than acquisition of a representation (long-term more than short-term memory). We sought to test whether these agents similarly impair learning in rats. METHODS: We used operant conditioning in rats to examine the effect of isoflurane on acquisition compared with long-term (24 h) memory of non-aversive olfactory memories using two different odour discrimination tasks. Rats learned the 'valences' of odour pairs presented either separately (task A) or simultaneously (task B), under control conditions and under isoflurane inhalation. In a separate set of experiments, we tested the ability of the animals to recall a learning set that had been acquired 24 h previously. RESULTS: Under 0.4% isoflurane inhalation, the average number of trials required to reach criterion performance (18 correct responses in 20 successive trials) increased from 21.9 to 43.5 (P<0.05) and 24.2 to 54.4 (P<0.05) for tasks A and B, respectively. Under 0.3% isoflurane inhalation, only task B was impaired (from 24.2 to 31.5 trials, P<0.05). Recall at 24 h was dose-dependently impaired or prevented by isoflurane for both tasks. CONCLUSIONS: Isoflurane interfered with long-term memory of odour valence without preventing its acquisition. This paradigm may serve as a non-aversive animal model of conscious amnesia.

Accelerated tissue aging and increased oxidative stress in broiler chickens fed allopurinol.

Uric acid has been hypothesized as being one of the more important antioxidants in limiting the accumulation of glycosylated endproducts in birds. Study 1 was designed to quantitatively manipulate the plasma concentrations of uric acid using hemin and allopurinol while study 2 determined their effects on skin pentosidine, the shear force value of Pectoralis major muscle, plasma glucose, body weight and chemiluminescence monitored oxidative stress in broiler chickens. Hemin was hypothesized to raise uric acid concentrations thereby lowering oxidative stress whereas allopurinol was hypothesized to lower uric acid concentrations and raise measures of oxidative stress. In study 1 feeding allopurinol (10 mg/kg body weight) to 8-week-old broiler chicks (n=50) for 10 days decreased plasma uric acid by 57%. However, hemin (10 mg/kg body weight) increased uric acid concentrations 20%. In study 2, 12-week-old broiler chicks (n=90) were randomly assigned to either an ad libitum (AL) diet or a diet restricted (DR) group. Each group was further divided into three treatments (control, allopurinol or hemin fed). Unexpectedly, hemin did not significantly effect uric acid concentrations but increased (P<0.05) measures of chemiluminescence dependent oxidative stress in both the DR and AL birds probably due to the ability of iron to generate oxygen radicals. Allopurinol lowered concentrations of uric acid and increased (P<0.05) the oxidative stress in the AL birds at week 22, reduced (P<0.05) body weight in both the AL and DR fed birds at 16 and 22 weeks of age, and markedly increased (P<0.001) shear force values of the pectoralis major muscle. Skin pentosidine levels increased (P<0.05) in AL birds fed allopurinol or hemin fed birds, but not in the diet restricted birds at 22 weeks. The significance of these studies is that concentrations of plasma uric acid can be related to measures of oxidative stress, which can be linked to tissue aging.

A characterization of amiodarone-induced pulmonary toxicity in F344 rats and identification of surfactant protein-D as a potential biomarker for the development of the toxicity.

Amiodarone (AD) is gaining support as a first-line antiarrhythmic drug despite its potentially fatal pulmonary toxicity involving inflammation and fibrosis. The goals of this study were to characterize a rat model of AD-induced pulmonary toxicity (AIPT) and identify a serum biomarker to aid in the diagnosis of the onset of pulmonary toxicity. Male F344 rats were instilled intratracheally with AD (6.25 mg/kg with a 3.125 mg/ml solution) in sterile water or the sterile water vehicle on days 0 and 2, a protocol that led to the development of pulmonary fibrosis on day 28 in the AD-treated animals. Animals were killed on days 3, 5, 6, 7, or 10 and bronchoalveolar lavage (BAL) was performed. Recovery of alveolar macrophages and eosinophils was increased on days 3 and 5, while neutrophil recovery and albumin levels in the first BAL fraction were significantly elevated only on day 3. BAL cells recovered from AD-treated rats at day 3 produced more phorbol myristate acetate-stimulated luminol-dependent chemiluminescence (LDCL) over 20 min than BAL cells from control rats. Experiments using specific inhibitors implicated superoxide and nitric oxide in at least part of the LDCL response. Serum levels of surfactant protein-D (SP-D), a surfactant-associated protein, were increased concurrently with the inflammatory response in the lungs. These findings indicate that this model exhibits transient pulmonary inflammation and damage, with the potential for elevated oxidant production in the lungs and subsequent pulmonary fibrosis. Also, SP-D is proposed as a specific biomarker to monitor the onset of AIPT in this model.

Green tea extract and its polyphenols markedly inhibit luminol-dependent chemiluminescence activated by peroxynitrite or SIN-1.

This study is based on a simple chemical interaction of peroxynitrite (OONO-) and luminol, which produces blue light upon oxidation. Since peroxynitrite has a half-life of less than 1 s, a drug known as SIN-1 is used as a peroxynitrite generator. In addition peroxynitrite itself was used directly with a fast injection-mixing system to ascertain whether there are differences between it and the peroxynitrite-generating system (SIN-1) which mimics the natural production of (OONO-). Peroxynitrite is a potent oxidizing compound (1000 times more active than equidose hydrogen peroxide) and it can oxidize carbohydrates, lipids, proteins and nucleic acids. Upon stimulation by inflammation and/or infection, macrophages and neutrophils can be activated to produce large amounts of peroxynitrite. We are interested in simple chemicals that are non-toxic that could inhibit or destroy peroxynitrite, which might otherwise cause inappropriate damage to blood and tissues. Green tea is a complex mixture containing several potent major antioxidant constituents, eg flavins and/or polyphenols. The constituents in green tea may react directly or indirectly with peroxynitrite or its constituents through the process of antioxidation to inhibit light. Alternatively, compounds could produce superoxide which, when reacted with nitric oxide, could produce more peroxynitrite and hence more light with luminol. Therefore, as the tea or antioxidants from tea are diluted, while the peroxynitrite or its precursors are kept at a constant concentration, one can observe unusual behaviour regarding light emission. Initially, at high doses of tea or antioxidant, one observes clear inhibition of the light generated from the reaction of peroxynitrite and luminol. However, at dilute concentrations of antioxidants, one can often observe stimulation of light. Possible reasons for these observations are discussed.

A new screening method to detect water-soluble antioxidants: acetaminophen (Tylenol) and other phenols react as antioxidants and destroy peroxynitrite-based luminol-dependent chemiluminescence.

This study is based on a simple chemical interaction of peroxynitrite (O = N-O-O-) and luminol, which produces blue light upon oxidation. Since peroxynitrite has a half-life of about 1 s, a drug known as linsidomine (SIN-1) is used as a peroxynitrite generator. Peroxynitrite can oxidize lipids, proteins and nucleic acids. Upon the stimulation of inflammation and/or infection, macrophages and neutrophils can be induced to produce large amounts of peroxynitrite, which can oxidize phenols and sulphhydryl-containing compounds. Therefore, phenols and sulphhydryls eliminate peroxynitrite. This is an example of the Yin-Yang hypothesis e.g. oxidation-reduction. Acetaminophen (Tylenol) can inhibit fever and some types of pain without being a particularly effective anti-inflammatory. Since it is a phenol, it could act as a nitration target for peroxynitrite. Then peroxynitrite, the possible cause of pain and elevated temperature, might be destroyed in the reaction. Acetaminophen is a phenolic compound which produces a clear inhibitory dose-response curve with peroxynitrite in its range of clinical effectiveness. Whether acetaminophen actually works as we suggest is to be proven. Three different types of reaction could decrease the amount of peroxynitrite: (a) interference with base-catalysed opening of the SIN-1 molecule; (b) destruction of one or both substances needed to form it--superoxide and/or nitric oxide; when the SIN-1 degrades to superoxide and nitric oxide, the former may be destroyed by superoxide dismutase (SOD); (c) peroxynitrite may react directly with phenols (mono-, di-, tri- and tetraphenols), possibly by nitration. Nordihydroguaiaretic acid and 2-hydroxyestradiol (catechol estrogen) are potent inhibitors of luminol light emission. Epineprine, isoproterenol, pyrogallol, catechol and ascorbic acid (a classic antioxidant) are all inhibitors of luminol chemiluminescence. Isoproterenol, norepinephrine/and epinephrine first inhibit light but overall stimulate the light production. Initially, SIN-1 degrades to produce peroxynitrite, which reacts with luminol to produce blue light. If any of three catecholamines are present with the reaction that produces light, there is an initial inhibition of light production, and then a marked stimulation. A possible reason for this is that these catechols are oxidized and the metabolized phenol stimulates the production of light from luminol. Also, during oxidation of catecholamines superoxide is sometimes formed, which could stimulate production of peroxynitrite. This simple screening system is introduced to find useful antioxidants against peroxynitrite.

Silica-induced pulmonary inflammation in rats: activation of NF-kappa B and its suppression by dexamethasone.

The goal of this study was to examine the relationship of the transcriptional regulatory factor nuclear factor-kappaB (NF-kappa B) to the early inflammatory events involved with silica exposure. Male F-344 rats received an intratracheal (i.t.) instillation of silica (100 mg/kg in a volume of 1 ml/kg) of saline. At 1, 3, 6, and 18 h postinstillation, and the rats were sacrificed and underwent bronchoalveolar lavage (BAL) for functional analysis of inflammation. Beginning at 1 h postinstillation, the silica-instilled (Si) rats displayed significant increases in neutrophils in BAL fluid compared to the saline controls. BAL cells from the Si group displayed a significant increase in luminol-dependent chemiluminescence (LDCL) compared to the controls. NF-kappa B activation was measurable at 3 h postinstillation, and this activation continued throughout the 18-h time course. Treatment with dexamethasone (5 mg/kg) at -3 h prior to silica instillation, at the time of instillation (0 h), and +1.5 h postinstillation resulted in both a reduction in NF-kappa B expression (by 70%) at 3 h postinstillation and corresponding reductions in LDCL, BAL cell count, and BAL neutrophils. These results show that activation of NF-kappa B is associated with silica-induced pulmonary inflammation, and the inhibition of its activation correlates temporally with suppression of inflammation.

The possible role of peroxynitrite in Alzheimer's disease: a simple hypothesis that could be tested more thoroughly.

Alzheimer's disease is characterized by the development of a degenerative condition in the elderly, associated with dementia. Upon pathological examination, cerebral amyloid plaques are found which contain denatured protein or peptide material. The process of denaturation of protein requires the presence of excessive heat, organic solvents, or oxidizing acids (OA). It seems that only OA could produce these effects since the other two are not present in the disease. Macrophages can produce the anion of an oxidizing acid known as peroxynitrite (OONO). This material is formed from two free radical gases, namely superoxide anion [.O2]- and nitric oxide (.N = O). Although (OONO)- is very reactive (1000 times more oxidizing than hydrogen peroxide), its half life in solution is only 1 to 2 seconds. Therefore, when it oxidizes a substance (such as protein) peroxynitrite disappears. The brain contains cells called microglia which are produced from monocytes in the same way as other types of macrophages from the lung and liver etc. The macrophages from the lung (alveolar) and liver (Kupfer cells) produce large amounts of peroxynitrite when activated by particles (silica) or infectious agents (lipopolysaccharide or interferon). Microglia produce highly oxidizing substances as well, but no one has ever measured production of peroxynitrite from these cells. Assuming that microglia produce peroxynitrite, or other similar oxidants, anti-oxidant and anti-inflammatory drugs should be helpful in treatment of early forms of the disease. In addition, large doses of anti-oxidant vitamin C and vitamin E might be helpful to people with Alzheimer's disease.

Characteristics of the acute-phase pulmonary response to silica in rats.

Exposure to silica, a cytotoxic and fibrogenic mineral dust, has been demonstrated to cause pulmonary inflammation and damage to the lung tissue. In contrast to the long-term consequences, little information exists on the sequence of inflammatory/damaging events occurring acutely after exposure to silica. The purpose of this study was to determine the minimum time after the administration of silica that the inflammatory/damage response is detectable and the temporal relationship of these processes. Male Fischer 344 rats were dosed intratracheally with silica (2.5 or 10 mg/100 g body weight) or saline vehicle. At 2 and 4 h after instillation, both cellular (total cell count and neutrophil count) and biochemical (total protein, albumin, and beta-glucuronidase and lactate dehydrogenase activities) parameters of inflammation and damage were evaluated in the bronchoalveolar lavage fluid. At 2 h, total protein levels were elevated at both silica doses, but all other parameters were unchanged; however, 4 h after silica exposure all parameters were elevated over those of the saline control. In a further attempt to characterize the inflammatory/damage processes, luminol-dependent chemiluminescence (LDCL) was performed on aliquots of chopped lung. At 2 h after silica instillation, phorbol myristate acetate-stimulated lung tissue from silica-treated rats had no increase in light production when compared to controls, whereas after 4 h there were significant increases in LDCL activity in both dose groups when compared to controls. The addition of superoxide dismutase (SOD) decreased LDCL activity of the 2.5 mg/100 g group by 59% (2 h) and 66% (4 h), and of the 10 mg/100 g group by 49% (2 h) and 73% (4 h). Alternatively, the addition of N-omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, decreased the 2.5 mg/100 g group by 52% (2 h) and 60% (4 h). The 10 mg/100 g group was decreased by 67% (2 h), but only exhibited a 12% reduction at 4 h. SOD and L-NAME also inhibited the background LDCL in saline-treated rats. These reductions in LDCL activity indicate that reactive oxygen and nitrogen species play a role in the acute phase pulmonary response from silica. The results of this study indicate that the initial stages of damage begin to appear by 2 h, but damage and inflammation are definitive by 4 h after administration of silica in rats.

Attenuation of acute inflammatory effects of silica in rat lung by 21-aminosteroid, U74389G.

Chemical alteration of the glucocorticoid, methylprednisolone, has led to the introduction of a new class of compounds called the 21-aminosteroids (21-ASs). The purpose of this study was to investigate the effect of the 21-AS, U74389G, on silica-induced acute lung injury. Male Fischer 344 rats were treated intraperitoneally with saline or U74389G in a total dose of 15 mg/kg divided into three injections of 5 mg/kg separated by 4 h. Following the first treatment, animals from the two groups were intratracheally instilled with silica (10 mg/100 g body wt in 0.5 ml of saline) or saline vehicle (0.5 ml). Twenty-four hours after the instillations, bronchoalveolar lavage (BAL) was performed. In the animals not receiving U74389G, marked increases in total protein, beta-glucuronidase, and lactate dehydrogenase (LDH) activities and number of neutrophils (PMNs) were demonstrated in the BAL fluid of the silica-treated animals compared to their controls. Silica also caused dramatic increases in the luminol-dependent chemiluminescence (CL) of lung tissue and BAL cells. The CL reaction was decreased by superoxide dismutase (SOD) and N-nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric oxide (NO) synthase inhibitor. In animals treated with U74389G, there was attenuation of the silica-induced increases in biochemical, cellular, and chemiluminescent indices of damage. This study demonstrates that U74389G significantly reduces acute lung injury caused by the intratracheal instillation of silica, and this drug may be of potential value for treatment of lung diseases in which damage caused by reactive oxygen species has been implicated.

Introduction of luminol-dependent chemiluminescence as a method to study silica inflammation in the tissue and phagocytic cells of rat lung.

The inhalation of silica has been shown to produce a dramatic inflammatory and toxic response within the lungs of humans and laboratory animals. A variety of cellular and biochemical parameters are used to assess the silica-induced lung injury. The purpose of this paper is to introduce the use of luminol-dependent chemiluminescence as a new method to study inflammation in both phagocytic cells and lung tissue recovered from silica-exposed animals. Chemiluminescence, or the emission of light, accompanies the release of reactive forms of oxygen when phagocytic cells are challenged. In this study, male Fischer 344 rats were intratracheally instilled with either silica (10 mg/100 g bw) or saline vehicle. One day after the instillations, a marked increase in the chemiluminescence was observed in the lung tissue and bronchoalveolar lavage cells recovered from the silica-treated animals when compared with the saline controls. The light reaction was markedly decreased by either superoxide dismutase of N-nitro-arginine methyl ester hydrochloride. Superoxide dismutase is involved in the enzymatic breakdown of superoxide anion, while N-nitro-L-arginine methyl ester hydrocholoride, a nitric oxide synthase inhibitor, prevents the formation of nitric oxide. When superoxide anion and nitric oxide react, they form the highly oxidizing substance peroxynitrite. This study then implicates peroxynitrite as an agent that may be responsible for some of the oxidant lung injury that is associated with silica exposure. The use of luminol-dependent chemiluminescence may prove valuable as a method to measure the earliest events in the inflammatory process, and may be an adjunct in studying the mechanisms that produce inflammation.

Interaction of artemisinin and tetracycline or erythromycin against Plasmodium falciparum in vitro.

Antimalarial activities of tetracycline (TC) and erythromycin (EM), alone or in combination with artemisinin (Qinghaosu, QHS), were studied using chloroquine (CQ)-sensitive (D6) and -resistant (W2) strains of Plasmodium falciparum in vitro. The antimalarial potency of TC (IC50 = 9862 nM for the CQ-sensitive parasite, 32414 nM for the CQ-resistant one) or EM (IC50 = 45787 nM for the CQ-sensitive parasite, 33397 nM for the CQ-resistant one) was much less than that of QHS (IC50 ranging from 25 to 40 nM). The CQ-resistant falciparum parasite displayed a cross-resistance to TC, while both the drug-sensitive and -resistant parasites exhibited similar responses to EM. However, antimalarial potency of EM appeared to be less than that of TC against the drug-sensitive parasite. When TC was combined with QHS, an additive interaction was observed against the CQ-sensitive falciparum parasite, while synergism was found with the CQ-resistant parasite. When EM was tested in combination with QHS, a potentiating interaction occurred with both the CQ-sensitive and resistant falciparum parasite. The above results indicated that the QHS combination with either TC or EM may be a promising antimalarial preparation with low recrudescence compared to artemisinin used alone in clinical practice.

The inhibition of silica-induced lung inflammation by dexamethasone as measured by bronchoalveolar lavage fluid parameters and peroxynitrite-dependent chemiluminescence.

The inhalation of silica has been shown to produce a dramatic inflammatory and toxic response within the lungs of humans and laboratory animals. Currently, no effective treatment exists for workers who may have been exposed to the inhalation of silica. The objective of this study was to develop an animal model in which we could evaluate the effect that anti-inflammatory steroids have on the acute silica-induced pulmonary inflammatory response. Male Fischer 344 rats were pretreated with either dexamethasone (2 mg/kg) or saline vehicle (i.p.) on days 1, 3, and 5. On day 6, the animals from the two groups were then intratracheally instilled with either silica (20 mg/0.5 ml saline vehicle) or saline vehicle (0.5 ml). Twenty-four hours after the instillations in the non-steroid group, significant increases occurred in total protein, total number of cells, neutrophils, and lymphocytes recovered from the lungs of animals treated with silica compared to saline controls. Silica also caused dramatic increases in the luminol-dependent chemiluminescence (LDCL) of lung tissue and bronchoalveolar lavage (BAL) cells. The LDCL reaction was markedly decreased by either superoxide dismutase (SOD) or N-nitro-L-arginine methyl ester hydrochloride (L-NAME). SOD is involved in the enzymatic breakdown of superoxide anion, while L-NAME, a nitric oxide (NO) synthase inhibitor, prevents the formation of NO. When the superoxide anion and NO react, they form the highly oxidizing substance peroxynitrite. This study then implicates peroxynitrite as an agent which may be involved in the silica-induced oxidant lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of chloroquine resistance in falciparum malaria by some calcium channel inhibitors and optical isomers is independent of calcium channel blockade.

Various types of calcium channel blockers verapamil, gallopamil, devapamil, diltiazem, and nifedipine and a calmodulin inhibitor R24571 were evaluated for reversal of chloroquine(CQ) resistance of Plasmodium falciparum in an in vitro system. The results demonstrated that some of the above Ca2+ antagonists such as verapamil, gallopamil, devapamil and diltiazem were found to exert remarkable reversal activity of CQ resistance of the falciparum parasite in vitro, while the others like nifedipine and R24571 had no reversal properties of CQ resistance of the parasite. In addition, reversal activities of the CQ resistance by enantiomers of some calcium channel blockers(R-(+)-verapamil, R-(+)-gallopamil and R-(+)-devapamil), which do not bind to the calcium channel, were also observed in this study. The data strongly indicate that the mechanism of reversal of CQ resistance of falciparum malaria in vitro is independent of the calcium channel.

Regulation of rat growth hormone gene expression in tissue culture: influence of cell growth phase.

Regions of the rat growth hormone gene (rGH) upstream of the transcription initiation site were cloned upstream of the firefly luciferase reporter gene to study promoter activity in GH3 rat pituitary cells during lag, logarithmic and plateau phase cell growth. The region -1751bp to -1329 activated gene expression in lag phase cells, but was neutral during logarithmic and plateau phase cell growth. Sequences between -1329bp and -553 were inhibitory during lag and plateau phase growth, but activated promoter activity when introduced into cells during logarithmic growth. We conclude that the rGH 5' flanking DNA provides an interesting model to study DNA sequences involved in growth-related changes in promoter activity.

Response of alveolar macrophages to in vitro exposure to freshly fractured versus aged silica dust: the ability of Prosil 28, an organosilane material, to coat silica and reduce its biological reactivity.

We have reported previously that crushing or grinding crystalline silica results in the generation of silica-based radicals on the particulate surface and that these radicals can generate hydroxyl radicals in aqueous solution. Data in the present study indicate that freshly ground silica is more cytotoxic and is a more potent activator of alveolar macrophages than comparably sized aged silica. That is, compared to aged silica, fresh silica is 4.2-fold more potent in decreasing the membrane integrity of macrophages; is 50% more potent in activating hydrogen peroxide secretion by macrophages; and is 4.6-fold more potent in stimulating cellular chemiluminescence. Prosil 28, an organosilane material, is an effective coating agent for fresh silica. It decreases the cytotoxicity of fresh silica by as much as 78% and decreases the ability of fresh silica to induce chemiluminescence from alveolar macrophages by 58%. The data suggest that surface radicals associated with freshly cleaved dust may be an important factor in the induction of pulmonary disease. Furthermore, treating dust with coating agents may substantially decrease toxicity.

The potentiating action of tetrandrine in combination with chloroquine or qinghaosu against chloroquine-sensitive and resistant falciparum malaria.

Using chloroquine-sensitive (CS) and chloroquine-resistant (CR) strains of Plasmodium falciparum in vitro, interactions between tetrandrine (TT) and either chloroquine (CQ) or qinghaosu (QHS, artemisinin) were assessed using isobolograms. Sums of the fractional inhibitory concentration for the combination of the two drugs are less than one and therefore, we can conclude that in vitro TT and CQ or QHS act synergistically against CS and CR falciparum malaria. Remarkably, using CR malaria, TT can lower the IC50 dose of CQ as much as 40 fold. These drug combinations may impair the advantage that the development of CQ resistance conveys on the parasite.

3H-azidopine photoaffinity labeling of high molecular weight proteins in chloroquine resistant falciparum malaria.

Using 3H-azidopine, we have succeeded in labeling proteins from chloroquine resistant (CR) human falciparum malaria parasites in the molecular weight range of 155-170 kd. Vinblastine does not compete, but azidopine blocks the labeling using 3H-azidopine. Relatively little or no labeling of the 155-170 kd protein is seen in the chloroquine sensitive strain using 3H-azidopine. Further competition can be seen with nicardipine and reserpine (71%) respectively and verapamil (61%), chloroquine (48%), quinacrine (56%), trifluoperazine (32%) and chlorpromazine (33%). We speculate that this may be the glycoprotein responsible for the resistance to chloroquine in falciparum malaria.

Selective antimalarial activity of tetrandrine against chloroquine resistant Plasmodium falciparum.

Antimalarial activity of tetrandrine was studied using a continuous in vitro culture of Plasmodium falciparum. Experimental results showed that tetrandrine has potent antimalarial effect on both chloroquine sensitive and resistant strains of Plasmodium falciparum. Interestingly, tetrandrine is about three times more potent against the chloroquine resistant strain than it is against the sensitive strain based on their IC50 values, which were 5.09 x 10(-7) M for the sensitive strain and 1.51 x 10(-7) M for the resistant strain. In addition, reversal experiments revealed that tetrandrine cannot reverse chloroquine-resistance, although it has verapamil-like, calcium-channel-blocker activity.

X-ray microanalysis of Plasmodium falciparum and infected red blood cells: effects of qinghaosu and chloroquine on potassium, sodium, and phosphorus composition.

Cryosections of human red blood cells infected by Plasmodium falciparum were analyzed by energy dispersive x-ray microanalysis to determine the elemental composition of the parasites and their red cell hosts separately. The effects of two antimalarial drugs, qinghaosu and chloroquine, on potassium, sodium, and phosphorus concentrations were studied. Malarial infection causes a decrease in potassium concentration and an increase in sodium concentration in the host red cells. The drastic change in the cation composition, however, occurs only in red cells infected by late stage parasites (late trophozoite and schizont). Red cells infected by early stage parasites (ring stage) show only small changes in sodium concentration. Furthermore, the noninfected red cells in parasitized cultures show no difference in composition from those of normal red cells. Treatment of the parasitized cultures with qinghaosu (10(-6) M) or chloroquine (10(-6) M) for 8 hr causes phosphorus concentration of both early and late parasites to decrease. An 8 hr treatment with qinghaosu also produces a reduction in potassium and an increase in sodium concentrations in early and late parasites. In contrast, 8 hr treatment with chloroquine only causes a change in the sodium and potassium concentrations of the late stage parasites and does not affect the early stage parasites.