K. Van Dyke

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.

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.

Studies concerning the mechanism of action of antimalarial drugs—: Inhibition of the incorporation of adenosine-8-3H into nucleic acids of Plasmodium berghei☆

Abstract A new system for the detection of the effects of antimalarial drugs on nucleic acids in Plasmodium berghei has been developed. Biochemical evidence for the incorporation of adenosine-8-3H into DNA and RNA of the parasite is presented with some preliminary conditions of incorporation. Dose-response curves established with the new system generally show inhibitory responses between 10−6M to 10−4M for quinine, quinacrine, ethidium bromide and between 10−4M to 10−3M for chloroquine. Quinacrine, quinine, chloroquine and ethidium have been reported to intercalate into the DNA helix based on physical and chemical evidence. DNA and RNA polymerase from E. coli are inhibited by these intercalating drugs and dose-response curves of inhibition by these drugs of adenosine incorporation are strikingly similar to doseresponse curves from isolated polymerase enzymes. The substantiation of the relative ineffectiveness of chloroquine as an antimalarial in P. berghei infections reveals the importance of defining antimalarial action on the basis of whole blood antimalarial activity.

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

Abstract Alzheimers 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 (·O 2 ] − and nitric oxide (⊎N=0). 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 Alzheimers disease.

Automated systems for the fluorometric determination of nucleic acids by the ethidium bromide technique

Abstract An automated analysis of DNA and RNA has been developed that is an extremely adaptable system for measurement of a wide range of nucleic acid concentrations. Conditions for the assessment of DNA in the presence of RNA have been achieved by employing an elevated temperature to remove RNA with RNase. A marked effect of temperature on the fluorescence of nucleic acids in the presence of ethidium bromide has been found, therefore necessitating control of temperature for the reproducible assessment of nucleic acids.

Effects of Eugenol on Polymorphonuclear Cell Migration and Chemiluminescence

In this study, the effects of eugenol on human polymorphonuclear (PMN) cell migration and chemiluminescence were examined in vitro. Utilizing zymosan-activated serum or crude Bacteroides sonicate fractions as chemotractants, we found that eugenol inhibits PMN migration at 6.6 x 10 -2 to 6.6 x 10 -5 mol/L (P<0.05). Also, similar effects were observed in PMNs pre-incubated in eugenol. Regardless of concentration, eugenol was not found to induce chemotaxis of PMNs. An examination of PMN membrane activation through chemiluminescence gave results consistent with the chemotaxis data, demonstrating a decrease in light emission at concentrations as low as 6.6 x 10-6 mol/L (P<0.05). In view of these data, the potential effect of eugenol on in vivo (sulcular or periapical) PMN function deserves further study.

The effect of plant growth substances and natural products on RNA and DNA synthesis in leukocytes

The effect of auxins, cytokinins, gibbrellins and phenolics on the incorporation of uridine and thymidine into the nucleic acids of human leukocytes was examined. Both the stimulation and inhibition of the incorporation of the precursors was noted. The auxins consistently promoted the incorporation of uridine.

Plasmodium berghei: In vitro incorporation of purine derivatives into nucleic acids☆

Abstract Uptake of radioactive adenosine by Plasmodium berghei parasitized erythrocytes is sensitive to competition for uptake over a wide range of concentrations of nonradioactive adenosine. Infection points in the uptake curve for adenosine-8- 3 H occur between 10 −4 to 10 −5 and 10 −9 to 10 −10 m nonradioactive adenosine. This finding prompted study of the optimal conditions for the in vitro incorporation of radioactivity from AMP-8- 3 H into DNA and RNA of erythrocyte-free parasites. These conditions include: incubation temperature (37C), pH (7.4), amount of tritiated precursor (2.5 μCi), and stability of free parasite preparations upon storage in the cold (up to 4 hr at 4C). Whether a deoxyribose (deoxyadenosine-8- 3 H) or ribose (adenosine-8- 3 H) form of purine nucleoside is used, radioactivity associates primarily with parasite RNA (95% of total incorporation) but only slightly with parasite DNA (5% of total incorporation). The ratio is similar for incorporation of radioactivity from other precursors (purine nucleotides: AMP-8- 3 H, ADP-8- 3 H, ATP-8- 3 H, GTP-8- 3 H). During the first 20 min of incubation, radioequivalent amounts (2.5 μCi) of exogenous adenine derivatives incorporate into parasite nucleic acids in decreasing order as follows: AMP-8- 3 H > ADP-8- 3 H > ATP-8- 3 H > adenosine-8- 3 H > adenine8- 3 H > cyclic-3′,5′-AMP-8- 3 H. No detectable incorporation into free parasite nucleic acids occurs when either ATP-α- 32 P or ATP-β-γ- 32 P is added as substrate. Incorporation into either RNA or DNA is greatest when exogenous AMP-8- 3 H is used as substrate. Incorporation of radioactivity from AMP-8- 3 H into nucleic acids of erythrocyte-free parasites is twofold greater than into nucleic acids of the parasitizedblood equivalent. Incorporation from AMP-8- 3 H into free parasite nucleic acids is sensitive to competition for incorporation by nonradioactive AMP (10 −5 m nonradioactive AMP decreases incorporation from AMP-8- 3 H by 54%; 10 −3 m nonradioactive AMP decreases incorporation from AMP-8- 3 H by 98%). Similarly, over a range of concentrations, 6-mercaptopurine (6-MP) competes for and decreases incorporation of label from exogenous AMP-8- 3 H.

Effect of Cancer Chemotherapeutic Agents on the Chemiluminescence of Human Granulocytes

21 unilateral breast cancer patients taking different combinations of chemotherapeutic agents (cyclophosphamide, methotrexate, 6-fluorouracil, vincristine, and prednisone) were studied to determine how chemotherapy affected their granulocytes. It is widely believed that in cancer patients chemotherapeutic agents increase susceptibility to infection. Therefore, luminol-enhanced chemiluminescence was used to evaluate leukocyte function since the chemiluminescence response has been correlated to bacterial killing. the chemiluminescence response in cancer patients (6-week treatment) was significantly reduced (approximately 50%; p less than 0.01) compared to nontreated volunteers. Preliminary studies using 3H-formyl-methionyl-leucyl-phenyl alanine binding showed similar decreases. We postulate that chemotherapy for 6 weeks may affect granulocyte precursor cells in bone marrow, thereby weakening peripheral granulocytes and reducing both their bactericidal capacity and 3H-formyl-methionyl-leucyl-phenyl alanine receptors.

Comparison of the interaction of tricyclic antidepressants with human polymorphonuclear leukocytes as monitored by the generation of chemiluminescence

The interation of imipramine with human polymorphonuclear leukocytes (PMNs) results in a chemiluminescence (CL) response which has been attributed to the electronic excitation of the imipramine molecule resulting from a reaction of the drug with reactive oxygen species. In order to determine what portion of the tricyclic molecule is involved in this reaction, the interaction of other tricyclics with PMNs was monitored by chemiluminescence. It was observed that tricyclic antidepressants having a carbon atom at position 5 of the ring moiety (amitriptyline, for example) did not yield CL with either resting or zymosan-activated PMNs. In fact this group of compounds inhibited the zymosan-induced CL response. However, CL was observed, with both resting and metabolically-activated PMNs, from several tricyclics having a heterocyclic nitrogen at position 5. These included imipramine, desipramine, opipramol and iprindole. Chlorimipramine, which has a chlorine atom at position 3 of the ring system, failed to yield CL with resting or stimulated cells. Similarly, imipramine N-oxide failed to yield CL with resting cells, but enhanced CL was observed with zymosan-activated PMNs. On the basis of these observations it appears that some aspect of the ring moiety, other than just a heterocyclic nitrogen, facilitates a reaction between these molecules and reactive oxygen which culminates in the generation of CL.