Proteins from human cerebrospinal fluid: binding with nucleic acids.

Cerebrospinal fluid (CSF) contains two groups of proteins that bind tightly to DNA and to polyriboguanylic acid, respectively. In certain diseases the amounts of a given nucleic acid bound by a constant volume of CSF may increase, while in others the amount of such proteins may be reduced. Binding of polyriboguanylic acid increased in CSF samples from patients with brain tumors, stroke, multiple sclerosis, and communicating hydrocephalus, but it significantly decreased in CSF samples from patients with obstructive hydrocephalus. These increases may or may not be proportional to the rise in total CSF proteins characteristic for these diseases. Elevated binding of DNA was observed in samples from patients with hydrocephalus, epilepsy, and cortical atrophy. The technique described may be applicable to the diagnosis of a variety of diseases of the central nervous system.

Carcinogen and microsomal membrane interactions: changes in membrane density and ability to bind nucleic acids.

DNA and synthetic copolymer polyribocytidylic-polyriboguanylic acid bind to microsomal membrane. The nucleic acid-membrane complex may be detected by centrifugation in cesium chloride density gradients. The density of the nucleic acid-membrane complex and, in certain cases, the amount of nucleic acid associated with the membrane was changed in the presence of various carcinogenic chemicals.

Increased attachment of nucleic acids to eukaryotic and prokaryotic cells induced by chemical and physical carcinogens and mutagens.

Significantly enhanced attachment to Ehrlich ascites and Escherichia coli cells was observed for radioactive DNA and RNA in the presence of chemical mutagens and ultimate carcinogens. In some instances, formation of nucleic acid-protein adducts by these compounds further (or similarly) enhanced the binding. DNA irradiated with ultraviolet light in the presence of a protein bound more efficiently than either an unirradiated mixture of these two macromolecules or DNA irradiated alone. The spectrum of compounds tested and found active in this system includes alkylating agents, aromatic amines, and carcinogenic metals. Precarcinogens and nonultimate carcinogenic chemicals, as well as tumor-promoting agents, did not increase the binding. However, addition of extracts from mouse or rat livers activated precarcinogenic and proximate carcinogenic chemicals and resulted in enhanced cellular attachment of indicator nucleic acids in their presence. Possible usefulness of this test system for fast and efficient screening for environmental carcinogens and mutagens, as well as possible relevance of the observed phenomena to in vivo effects of chemical and physical carcinogens, is considered.

Macromolecular complexes produced by 1,3-propanesultone.

1,3-Propanesultone produces in vitro complexes between DNA and DNA, DNA and RNA, RNA, and RNA, DNA and proteins, and possibly RNA and proteins. All these interactions were detected with a variety of independent analytical techniques. Increased attachment of the 1,3-propanesultone-treated DNA to the microsomal membrane and enhanced adsorption to and penetration into Ehrlich ascites tumor cells were also observed. 1,3-Propanesultone was the second small-ring alkylating carcinogen studied in this laboratory that was found to produce such macromolecular complexes. The possibility that these highly diverse structures may be involved in chemical mutagenesis and tumorigenesis is considered.

Hereditary alterations in tumor-host relationship.

An indirect exposure of mice during their fetal stage of development to Ehrlich ascites tumor cells alters the animals' response to this tumor during their adult life. Control mice injected intraperitoneally with Ehrlich tumor cells react in various ways to the presence of this alien tissue. The response is especially evident during the first 48-72 h post-injection. Several aspects of this complex reaction are absent in those animals which were exposed to this tumor in utero. The measurably altered reactivity toward the Ehrlich tumor cells is hereditary. Possible mechanisms of this alteration are discussed.

Proximity of polyriboguanylic acid-reactive sites in mammalian DNA to portions of DNA preferentially hybridizing with cellular RNA.

Transcribed portions of mouse nuclear DNA are adjacent to sites reacting preferentially with polyriboguanylic acid [poly(G)]. This spatial relationship was suggested by hybridization of radioactively labeled Ehrlich ascites RNA with denatured mouse DNA, the latter fractionated by centrifugation in CsCl equilibrium density gradients both in the presence and in the absence of various synthetic polyribonucleotides. The fractions of DNA which preferentially reacted with guanylic acid-rich polyribonucleotides hybridized with radioactive RNA to a higher degree than did the bulk of cellular DNA. This ability to react with polyriboguanylic acid-containing polymers was further enhanced by breaking DNA by sonication. No increased tendency for such hybridization was seen with the DNA that preferentially reacted with polyribouridylic acid.

Intermolecular linking and fragmentation of DNA by beta-propiolactone, a monoalkylating carcinogen.

Brief exposure to beta-propiolactone (BPL) increases the sedimentation rate of purified Escherichia coli DNA in neutral and alkaline sucrose gradients. However, when electrophoresed in polyacrylamide-agarose gels, this BPL-treated DNA moves ahead of the control. Longer incubation with BPL gives rise to two new fractions, the first one sedimenting as a heterogeneous material of 6-8S, and the second one of very high sedimentation velocity. In acrylamide-agarose gels, the first fraction is again recovered in the 6-8S area, while the second fraction does not enter the gel at all. The DNA at this stage is hyperchromic in ultraviolet light suggesting that as much as 20% may be denaturated. Coliphage lambda DNA treated briefly with BPL and spread in a protein monolayer appears under the electron microscope as a rigid, extended molecule, up to 15% longer than the control DNA, and usually in compact, folded configurations suggesting intramolecular linking. After longer exposure, localized denaturation associated with single-strand breaks is observed. The single-stranded "whiskers" then interact with other DNA molecules, creating highly complex branched networks of single- and multi-stranded DNA. The possible relevance of these observations to the mechanisms involved in carcinogenesis and mutagenesis is considered.

DNA-cell-binding (DCB) assay for suspected carcinogens and mutagens.

This report describes a novel technique for screening potential carcinogens and mutagens. The DNA-cell-binding (DCB) assay is based on earlier observations which indicated that DNA and other nucleic acids exposed to active carcinogens strongly react with other macromolecules, producing nucleic acid--nucleic acid and nucleic acid--protein adducts. The latter group of adducts included complexes with proteins present in both prokaryotic and eukaryotic cell membranes. Increased attachment of DNA to the intact bacterial and animal cells was seen in the presence of active carcinogens or carcinogens activated by extracts from mouse and rat livers. We have conducted a survey of almost 280 chemicals including 130 with known carcinogenic potential (i.e., either known carcinogens or known non-carcinogens). The DCB test and animal assays agreed in abut 96% of cases. Thus, as a predictor of potential carcinogenicity, this assay compares favorably with other rapid methods currently in use. In this respect, the DCB assay is also superior to other techniques which measure the formation of macromolecular complexes, such as velocity centrifugation through sucrose gradients, gel electrophoresis, filtration through nitrocellulose filters, chromatography on methyl-esterified albumin, equilibrium density gradient centrifugation, etc. In the few cases for which the data were available, combining the results of DCB assays with the results of experiments in which the induction of DNA--protein adducts in living human cells in tissue culture has been measured by cold phenol extraction, the predictability was increased to 100%. We suggest that DCB assay should be used either alone or in combination with other rapid methods of carcinogen detection for screening industrial, environmental and other chemicals and chemical mixtures for their carcinogenic potential. Ways of further improving and simplifying the DCB tests are considered.

Alterations in Bacillus subtilis transforming DNA induced by beta-propiolactone and 1,3-propane sultone, two mutagenic and carcinogenic alkylating agents.

Transforming DNA was exposed to either beta-propiolactone or 1,3-propane sultone and then used for transformation of competent bacteria to nutritional independence from tyrosine and tryptophan (linked markers) and leucine (an unlinked marker). The ability to transform was progressively lost by the DNA during incubation with either of these two chemicals. For all three markers the inactivation curve was biphasic, with a short period of rapid inactivation followed by one characterized by a much slower rate. The overall rate of inactivation was different for all three markers and presumably was related to the size of the marker. The decrease in the transforming activity was in part due to the slower rate of penetration of alkylated DNA through the cellular membrane and its inability to enter the recipient bacteria. This decrease in the rate of cellular uptake, even for DNA eventually destined to enter the cell, began almost immediately after its exposure to the chemical and ended up with an almost complete lack of recognition of the heavily alkylated DNA by the specific surface receptors of competent cells. Such DNA attached to sites on the surface of competent bacteria which were different from receptors specific for the untreated nucleic acid. This attachment was not followed by uptake of the altered DNA. Presence of albumin during the incubation with a carcinogen further increased the degree of inactivation, indicating that the artificial nucleoproteins produced under such conditions were less efficient in the transformation assay than was the naked DNA. Cotransfomration of close markers progressively decreased, beginning immediately after the start of incubation of DNA with the chemicals. Extensively alkylated DNA fractionated by sedimentation through sucrose density gradients showed a peculiar distribution of cotransforming activity for such markers; namely, molecules larger than the bulk of DNA ("megamolecules") showed less ability to transform the second marker than did some of the apparently smaller molecules which sedimented more slowly through the gradient. An increase in cotransformation of distant markers was evident in DNA molecules after a short exposure to an alkylating agent, but cotransformation of such markers was absent in DNA treated for longer periods. The observed changes in the transforming and cotransforming activities of the alkylated DNA can be explained by what is known about the physicochemistry of such DNA and in particular about the propensity of the alkylated and broken molecules to form complexes with themselves and with other macromolecules.

Increased cotransformation of distant markers and altered patterns of DNA-cell interactions following the exposure of transforming DNA to two carcinogenic and mutagenic alkylating agents, diethyl and dimethyl sulfate.

Transforming DNA exposed to either diethyl sulfate (diES) or dimethyl sulfate (diMS) is inactivated. The rate of inactivation depends on the marker tested and on the chemical used: diMS is more active than diES. Cotransformation of linked markers is similarly depressed. In contrast, there is a transient increase in the cotransformation of distant, unlinked markers. These observations indicate that some of the intermolecular complexes of transforming DNA created in the test tube by the treatment with diES and diMS are biologically active. Radioactively labeled DNA treated with diES or diMS changes its patterns of interaction with cellular surfaces that are characteristic of untreated DNA. A possibility is considered that such alterations in DNA-protein interactions as well as the ability of these alkylating agents to transpose fragments of chromosomal material may play an important role in the processes of mutagenesis and, especially, carcinogenesis.

Potential toxicity of materials used for home insulation.

The two aqueous solutions used for production of residential home insulation by the so-called urea-formaldehyde process were tested for their ability to react with cellular macromolecules. One of the components (the catalyst-surfactant) changed the apparent molecular weight of isolated DNA and increased its rate of attachment to bacterial and animal cells. The other component (the formaldehyde-urea resin) showed both these activities, especially following its exposure to mouse or rat liver extracts (postmitochondrial supernatants). Actively growing HeLa cells exposed to the catalyst-surfactant solution and then extracted with phenol yielded diminishing amounts of DNA, suggesting the formation of strong bonds to other cellular macromolecules, most likely to proteins. Formation of complexes between nucleic acids and proteins, enhanced cellular binding of DNA, and decreased extractability of DNA from growing cells exposed to chemicals have been found in separate studies to correlate with carcinogenic activity of various substances. Since a significant number of buildings will be insulated with this urea-formaldehyde foam and since such foam is also used in agriculture on crops, appropriate precautions should be taken to limit human exposure to the component materials.

Formation of macromolecular complexes and other effects of DNA treatment with diethyl and dimethyl sulfate: physico-chemical and electron microscopic studies.

In vitro exposures of isolated DNA to one of the two carcinogenic and mutagenic chemicals, diethylsulfate or dimethylsulfate, induces several kinds of physicochemical and morphological alterations. These changes are detectable by a variety of independent techniques. A fraction of DNA treated briefly with either of these two chemicals moves during velocity centrifugation experiments less rapidly than the bulk of control DNA and more rapidly through gels during electrophoresis. This apparent decrease in size is paralleled by the formation of large DNA aggregates with mobilities indicating molecular weights several times that of the untreated, control DNA. The presence of a basic protein in the incubation mixture increases the rate of formation of such complexes. the tendency of the alkylated DNA to bind to both biological and non-biological materials is reflected in the increased attachment of DNA to columns built with methyl-esterified serum albumin and in its quantitative retention on nitrocellulose filters. DNA exposed to dimethylsulfate decreases its density in CsCl gradients. A mixture of two or more DNAs of different densities exposed to this chemical produces an u.v.-absorbing band which is found in such gradients at an intermediate density. If the alkylation reaction is carried out in the presence of a protein, a portion of DNA bands at a density intermediate between the density of DNA and that of the protein, even in the presence of an ionic detergent in the gradient. Under the electron microscope the alkylated DNA shows multiple single-strand breaks and peeling-off whiskers of denatured DNA. Aggregates of DNA molecules become visible upon further incubation of DNA with the alkylating agent. We suggest that the DNA-DNA and DNA- protein complexes play an important role in the process of carcinogenesis and mutagenesis.