William T. Speck

In Vitro Studies of Effects of Light and Riboflavin on DNA and HeLa Cells

Extract: The widespread use of phototherapy for neonatal hyperbilirubinemia has caused some concern since substances other than bilirubin may be photoactivated. The toxic properties of these photoactivated substances might prove to be more harmful to the neonatal infant than bilirubin. The purpose of the present study was to investigate the effect of photoactivated riboflavin in low concentration, on purified DNA. The results demonstrate extensive changes in the structure of DNA (see Table 1) manifested by (1) a change in the peak absorbance in the ultraviolet, (2) a decrease in the temperature of the thermal helix-coil transition, (3) a slight decrease in the sedimentation coefficient, and (4) an increase in the buoyant density values (1.704–1.709). These observations are consistent with the interpretation that there has been an alteration of one of the base moieties with minimal cleavage of the phosphodiester linkages. Results with human cells in tissue culture indicate that a similar photodynamic effect of riboflavin on the DNA occurs in living cells.Speculation: In view of the known relation between the ability of a variety of agents to modify DNA on the one hand and the potential to induce mutation and cancer on the other, the present results suggest that riboflavin supplementation as an adjunct to phototherapy for hyperbilirubinemia requires more thorough evaluation.

Comparative mutagenicity of aliphatic epoxides in Salmonella.

37 aliphatic epoxides comprising 6 subclasses (unsubstituted aliphatic epoxides, halogenated aliphatic epoxides, glycidyl esters, glycidates, glycidyl ethers and diglycidyl ethers) were tested, under code, for mutagenicity in Salmonella strains TA98, TA100, TA1535 and TA1537 and/or TA97 with and without metabolic activation using a standardized protocol. The 4 halogenated aliphatic epoxides and the 4 diglycidyl ethers were all mutagenic. The 2 glycidates were negative in all strain/activation systems used while all 5 glycidyl esters were mutagenic. 3 of the 8 unsubstituted aliphatic epoxides and 11 of the 12 glycidyl ethers were mutagenic. Glycidol also was mutagenic whereas 9,10-epoxyoctadecanoic acid, 2-ethylhexyl ester was not mutagenic. Of the 28 mutagenic compounds, all but neodecanoic acid, 2,3-epoxypropyl ester and 2-ethylhexyl glycidyl ether were detected in TA100 without activation. The latter two were detected only with activation in TA100 and TA1535. The majority of the other 26 chemicals were also mutagenic in TA1535 without activation. Good intra- and interlaboratory reproducibility was seen in the results of each of the 4 chemicals tested in more than one set of experiments. The current results confirm and extend the observations of other investigators regarding structural effects on the mutagenicity of members of the aliphatic epoxide class of chemicals.

Phototherapy for neonatal jaundice: Optimal wavelengths of light

Phototherapy results in transformation of bilirubin to more water-soluble isomers. The efficacy of monochromatic visible light from 350 to 550 nm in the fastest photoisomerization reaction was quantitated by high-pressure liquid chromatography. The most effective wavelengths in vitro (i.e., leading to greater than 25% photoisomer) were in the blue spectrum from approximately 390 to 470 nm. Green light (530 nm) was not only ineffective for production of photoisomer, but capable of reversing the reaction. The results indicate that any clinically useful phototherapy unit must include the blue portion of the visible spectrum, and suggest that the effectiveness of phototherapy may be increased by elimination of green light.

Phototherapy for neonatal jaundice: in vivo clearance of bilirubin photoproducts.

ABSTRACT: Phototherapy results in the conversion of native bilirubin to more water-soluble configurational and structural isomers. The serum half-life for the configurational isomer, the principal photoproduct in vivo, was determined by high pressure liquid chromatography in six premature infants following cessation of phototherapy. The mean half-life for this isomer was 15 h. The excretion of this isomer, calculated from the measured half-life, is less than half of daily bilirubin production, and therefore cannot account for the total bilirubin elimination observed during phototherapy. The serum concentration of the structural isomer, lumirubin, is lower than that of the configurational isomer; however, excretion is more rapid (serum half-life < 2 h). Because of its rapid excretion, lumirubin may be an important pathway for bilirubin elimination during phototherapy.

Mutagenicity testing of agent orange components and related chemicals.

Components of the herbicide Agent Orange--2,4-dichlorophenoxyacetic acid (2,4,-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and their esters, and the contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)--and related chemicals were tested for mutagenicity using Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537. No mutagenic activity was observed for any of the chemicals tested.

Proflavin: an unusual mutagen

In the Salmonella typhimurium mutagenicity assay, proflavin (PF), in the dark, was shown to possess some direct-acting frameshift mutagenic activity for strains TA1537 and TA98. In the presence of microsomal enzymes from mouse, hamster and rat livers, frameshift mutagenic activity for strain TA1538 and enhanced mutagenicity for strain TA1537 was observed. Exposure of bacteria to PF in the presence of visible light resulted in base substitution (strain TA1535) as well as frameshift (TA1537, TA1538, TA98) genetic activities. The findings are taken to indicate that PF possesses direct-acting frameshift activity (TA1537 and TA98) by virtue of its ability to intercalate between DNA base-pairs. The frameshift activity (TA1538, TA98 and enhanced TA1537) seen following metabolic or photo-activation may be due to the formation of chemical adducts between the cellular DNA and oxidation products of PF (a base-displacement effect). The light-induced base-substitution activity probably results from the generation of singlet oxygen which reacts with the guanine moiety of DNA. The mutagenic photoproduct of PF appears to be stable.

Phototherapy for Neonatal Jaundice: in Vitro Comparison of Light Sources

Summary: Phototherapy results in the conversion of bilirubin to more water-soluble isomers. Six clinically used phototherapy lamps which differ in their emission spectra have been compared in their ability to produce configurational and structural isomers of bilirubin in vitro. For all of the lamps, the initial rate of configurational isomerization was highly correlated (r = 0.969) with the intensity of irradiation falling within the bilirubin absorption band. The percentage of the total bilirubin converted to the configurational isomer at equilibrium was dependent upon the spectral distribution of the lamp, and was greatest (26.2 ± 1.3%) with the special blue lamp, which has a narrow spectral output centered at 445 nm. The rate of formation of the structural isomer, lumirubin, was generally dependent upon the intensity of irradiation within the bilirubin absorption band.

Short communication. Photochemical reactions of riboflavin: covalent binding to DNA and to poly (dA) . poly (dT).

Summary: Standard phototherapy illumination of human cells in the presence of riboflavin is known to produce single-strand breaks in intracellular DNA. A new photochemical reaction is described between riboflavin and purified DNA in which an adduct is formed. Unlike the previously described oxygen-dependent reaction between light-activated riboflavin and deoxyguanosine, this new photochemical reaction is oxygen-independent and involves deoxyadenosine or thymidine.

Mutagenicity and DNA-modifying activity of 2-nitropropane

The results presented herein confirm the mutagenicity in Salmonella of 2-nitropropane and the relative inactivity of 1-nitropropane. The lack of mutagenicity of 1- and 2-aminopropane is also reported. In addition, we have demonstrated the in vitro DNA-modifying activity of 2-nitropropane, and present evidence to suggest that genetic activity of this nitroalkane may not be dependent on the enzymic reduction of the nitro function to the corresponding hydroxylamine.

Intracellular Deoxyribonucleic Acid-modifying Activity of Phototherapy Lights

Extract: Little information is available on the intracellular consequences of the high intensity illumination with visible light that is commonly used in the treatment of neonatal hyperbilirubinemia. The present study was undertaken to determine whether DNA isolated from human cells growing in culture had undergone structural alterations as a result of exposure to high intensity visible light in the absence of added photosensitizers. Analysis of such DNA on alkaline sucrose gradients revealed a diminution in size after illumination. This structural lesion was repairable when the treated cells were subsequently incubated in the dark. These changes were observed at wave lengths (450 nm) of light identical with those utilized in phototherapy and with total light doses (70.4 kJ/m2) representing only 5% of that received by a newborn infant undergoing phototherapy in our nursery for a 24-hr period (1.3 × 103 kJ/m2).Speculation: In view of the known relationship between DNA-modifying activity and mutagenic and/or carcinogenic potential, the present results suggest that phototherapy is a complex process with an inherent potential for serious long term sequelae.