Relationship between acute and chronic exposures in mutagenicity studies in mice

The relationship between acute and chronic exposures in mutagenicity studies on mammals still lacks experimental data that might permit the decision whether or not the long-term exposures are of significance in mutagenicity testing.Fractional application of TEPA, THIOTEPA, EMS cyclophosphamide and sodium arsenite was made in experiments with mice, by using the dominant-lethal test and cytogenetic analysis of bone marrow. In most experiments the repeated application yielded the same or higher genetic injury than the same total dose at a single application. Negative results are discussed in relation to the threshold dose and the different sensitivity of the germ-cell stage.Possible interaction of mutagens was also studied by analyzing the combined effect of a long-term exposure to sodium arsenite, which probably affected the repair mechanism, and of a single dose of TEPA. It is concluded that the present stage of knowledge requires acceptance of the opinion that the genetic risk induced by chronic exposure to a chemical is as serious as that induced by an acute exposure.     

On the decarboxylation of α-keto acids by isolated chloroplasts

The mechanism of the decarboxylation of α-keto acids by isolated chloroplasts has been studied with the aid of superoxide dismutase and catalase. Using photosynthetic and enzymatic systems, which are known to catalyze peroxidic oxidations, we have been able to demonstrate that both the superoxide free radical ion and H₂O₂ are necessary for maximal rates of decarboxylation. In isolated chloroplasts, an auto-oxidizable electron acceptor as well as an electron donor for Photosystem I are absolute requirements for the decarboxylation. H₂O₂ seems to be the primary oxidant in the decarboxylation of pyruvate or glyoxylate by isolated chloroplasts. A secondary rate of decarboxylation is superimposed on the primary one, mediated by superoxide free radical ion. Mn²⁺ stimulates the decarboxylation probably via intermediarily-formed Mn³⁺ in a reaction, which is neither inhibited by catalase nor by superoxide dismutase. A decarboxylation of pyruvate or glyoxylate by isolated chloroplasts in the presence of NADP⁺ is initiated, as soon as the available NADP⁺ is fully reduced. In this case, the open-chain electron transport seems to switch from NADP⁺ to oxygen as the terminal electron acceptor.     

Thiono compounds. 4. In vitro mutagenic and antineoplastic activity of TEPA and thio-TEPA

Tris (1-aziridinyl) phosphine oxide (TEPA) and tris (1-aziridinyl) phosphine sulfide (thio-TEPA) induced base pair mutations in the Ames mutagenic assay. Thio-TEPA required metabolic activation while TEPA was active without metabolic activation. Growth of a human vaginal carcinoma (A431), a human breast carcinoma (MDAMB-231), and a human cervical carcinoma (HeLa) were inhibited in soft agar in vitro at concentrations which induced mutagenesis in the Ames Assay. A fourth line, JEG choriocarcinoma, was sensitive to the antigrowth properties of both drugs at concentrations below that which induced mutagenesis. These data suggest that as more antineoplastic agents become available, and as mean survival times increase, knowledge of the relative in vitro sensitivity of a patient's neoplasm to a specific antineoplastic drug (i.e., dose required for growth inhibition) as a function of its mutagenic index might be useful for prediction of clinical remission, as well as the risk of secondary neoplasm induction.Abbreviations TEPA tris (1-aziridinyl) phosphine oxide - thio-TEPA tris (1-aziridinyl) phosphine sulfide - MEM Minimal Essential Media This study was supported by PHS grant No. CA 30321 awarded by the National Cancer Institute (DHHS).     

Induction of translocations by cyclophosphamide in different germ cell stages of male mice: cytological characterization and transmission.

Cytological and fertility tests were performed in F1 male mice derived from different germ-cell stages of male parents treated with cyclophosphamide (350 mg/kg body weight). The objectives of the present experiment were: (I) to determine the sensitivity of the male germ-cell stages to the induction of translocations by the compound, and (2) to characterize translocation configurations in F1 and F2 males, in order to obtain information about the pattern of chromosome breakage induced and its transmission to subsequent generations. Of 508 F1 males studied, 39 were partially sterile. The group of males conceived 8-21 days after treatment contained by far the highest proportion of partially sterile animals (30%). It was also the only group in which totally sterile animals (11%) were found. Of 25 semisterile males from this group, 24 gave evidence of translocations when spermatocytes were scored at diakinesis. The translocation frequencies in F1 derived from treated spermatozoa and spermatocytes were 14 and 1%, respectively. No translocations were detected cytologically in 6 semisterile males derived from treated spermatogonial stages. These results indicate that spermatid stages are especially sensitive to the mutagenic action of cyclophosphamide. In 21 of the 31 semisterile translocation males (68%), the majority of the spermatocytes contained 18 bivalents plus a ring-of-four configuration, indicating that both breakpoints were relatively centrally located; and in several of these males, the frequency of cells with rings was close to 100%. In another 9 F1 males (29%) the predominant multivalent configuration was a chain-of-four, indicating one of the breakpoints to be relatively more terminally located; and in one male (3%), the majority of cells had two unequal bivalents, indicating both breakpoints to be fairly close to the ends of the chromosomes involved. Determination of centromere positions by the use of C-banding showed that chain-of-four configurations in any one male were predominantly of a given type..     

Energetics of the one-electron steps in the NAD+/NADH redox couple

The one-electron reduction potential of NAD⁺ has been determined using pulse radiolysis to study electron-transfer equilibria between it and a low potential bipyridylium compound. The determined value E¹₇ (NAD⁺/NAD^.) = ?922 ± 8 mV (NHE scale) is used to calculate E²₇ (NAD^./NADH) = +282 mV. E¹₇ for 1-methylnicotinamide, E¹₇ (MeN⁺/MeN^.) = ?918 ± 7 mV.     

Immobilization of <Emphasis Type="Bold">β</Emphasis>-fructofuranosidase from <Emphasis Type="Italic">Aspergillus japonicus</Emphasis> on chitosan using tris(hydroxymethyl)phosphine or glutaraldehyde as a coupling agent

A partially purified -fructofuranosidase from Aspergillus japonicus was covalently immobilized on to chitosan beads using either glutaraldehyde or tris(hydroxymethyl)phosphine (THP) as a coupling agent. Compared with the glutaraldehyde-immobilized and the free enzyme, the THP-immobilized enzyme had the highest thermal stability with 78% activity retained after 12 days at 37 ° C. The THP-immobilized enzyme also had higher reusability than that immobilized by glutaraldehyde, 75% activity was retained after 11 batches (or 11 days) at 37° C for the THP immobilized enzyme system. Less yield (48%) of fructooligosaccharides (FOS) were produced by the THP-immobilized enzyme compared with the free enzyme system (58%) from 50 (w/v) sucrose at 50 ° C.     

A one microsecond component of chlorophyll luminescence suggesting a primary acceptor of system II of photosynthesis different from Q

The kinetics of the luminescence of chlorophyll a in Chlorella vulgaris were studied in the time range from 0.2 μs to 20 μs after a short saturating flash ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 25}\text{ns}$) under various pretreatment including anaerobiosis, flashes, continuous illumination and various additions. A 1 μs luminescence component probably originating from System II was found of which the relative amplitude was maximum under anaerobic conditions for reaction centers in the state SPQ^? before the flash, about one third for centers in the state S⁺PQ^? or SPQ before the flash, and about one tenth for centers in the state S⁺PQ before the flash. S is the secondary donor complex with zero charge; S⁺ is the secondary donor complex with 1 to 3 positive charges; P, the primary donor, is the photoactive chlorophyll a, P-680, of reaction center 2; Q^? is the reduced acceptor of System II, Q. Under aerobic conditions, where an endogenous quencher presumably was active, the luminescence was reduced by a factor two.The 1 μs decay of the luminescence is probably caused by the disappearance of P⁺ formed in the laser flash according to the reaction ZP⁺ → Z⁺P in which Z is the molecule which donates an electron to P⁺ and which is part of S. After addition of hydroxylamine, the 1 μs luminescence component changed with the incubation time exponentially (τ = 27 s) into a 30 μs component; during the same time, the variable fluorescence yield, measured 9 μs after the laser flash, decreased by a factor 2 with the same time constant. Hereafter in a second much slower phase the fluorescence yield decreased as an exponential function of the incubation time to about the dark value; meanwhile the 30 μs luminescence increased about 50% with the same time constant (τ = 7 min). Heat treatment abolished both luminescence components.The 1 μs luminescence component saturated at about the same energy as the System II fluorescence yield 60 μs after the laser flash and as the slower luminescence components. From the observation that the amplitude is maximum if the laser flash is given when the fluorescence yield is high after prolonged anaerobic conditions (state SQ^?), we conclude that the 1 μs luminescence is probably caused by the reaction

$\text{PWQ}{}^{\mathrm{?}}\text{+hv →}\text{P}\text{1}\text{WQ}{}^{\mathrm{?}}\text{→}\text{P}{}^{\mathrm{+}}\text{W}{}^{\mathrm{?}}\text{Q}{}^{\mathrm{?}}\text{→}\text{P}\text{1}\text{WQ}{}^{\mathrm{?}}\text{→ PWQ}{}^{\mathrm{?}}\text{+hv}$

in which W is an acceptor different from Q. The presence of S⁺ reduced the luminescence amplitude to about one third. Two models are discussed, one with W as an intermediate between P and Q and another, which gives the best interpretation, with W on a side path.     

Identification of the 120 μs phase in the decay of delayed fluorescence in spinach chloroplasts and subchloroplast particles as the intrinsic back reaction. The dependence of the level of this phase on the thylakoids internal pH

After a 500 μs laser flash a 120 μs phase in the decay of delayed fluorescence is visible under a variety of circumstances in spinach chloroplasts and subchloroplast particles enriched in Photosystem II prepared by means of digitonin. The level of this phase is high in the case of inhibition of oxygen evolution at the donor side of Photosystem II. Comparison with the results of Babcock and Sauer (1975) Biochim. Biophys. Acta 376, 329–344, indicates that their EPR signal II_f which they suppose to be due to Z⁺, the oxidized first secondary donor of Photosystem II, is well correlated with a large amplitude of our 120 μs phase. We explain our 120 μs phase by the intrinsic back reaction of the excited reaction center in the presence of Z⁺, as predicted by Van Gorkom and Donze (1973) Photochem. Photobiol. 17, 333–342. The redox state of Z⁺ is dependent on the internal pH of the thylakoids. The results on the effect of pH in the μs region are compared with those obtained in the ms region.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment

A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O₂ evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.     

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

Guanosine 3',5'-monophosphate and the action of alkylating agents.

The intracellular level of guanosine 3',5'-monophosphate (cGMP) has been measured in Walker carcinoma cells in tissue culture after treatment with various alkylating agents. At concentrations which caused a rise in the level of adenosine 3',5'-monophosphate (cAMP) chlorambucil and 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954) produced only a small (35%) elevation of cGMP, while merophan had no such effect. This suggests that any effect of cAMP will not be outweighed by an equivalent rise in cGMP. Sepcific cytosolic binding of cGMP decreased with increasing resistance of Walker cells to alkylating agents, while the dissociation constant, KD, for binding increased. This was also observed with cAMP binding which suggests that the same protein in responsible for binding both nucleotides.     

pH dependent changes in the reactivity of the primary electron acceptor of System II in spinach chloroplasts to external oxidant and reductant

Changes in the rates of dark oxidation and reduction of the primary electron acceptor of System II by added oxidant and reductant were investigated by measuring the induction of chlorophyll fluorescence under moderate actinic light in 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-inhibited chloroplasts at pH values between 3.6 and 9.5. It was found that:

1. (1) The rate of dark oxidation of photoreduced primary acceptor was very slow at all the pH values tested without added electron acceptor.

2. (2) The rate was accelerated by the addition of ferricyanide in the whole pH range. It was dependent approximately on the 0.8th power of the ferricyanide concentration.

3. (3) The rate constant for the oxidation of the primary acceptor by ferricyanide was pH-dependent and became high at low pH. The value at pH 3.6 was more than 100 times that at pH 7.8.

4. (4) The pH-dependent change in the rate constant was almost reversible when the chloroplasts were suspended at the original pH after a large pH change (acid treatment).

5. (5) An addition of carbonylcyanide m-chlorophenylhydrazone or heavy metal chelators had little effect on the rate of dark oxidation of the primary acceptor by ferricyanide.

6. (6) The dark reduction of the primary acceptor by sodium dithionite also became faster at low pH.

From these results it is concluded that at low pH the primary acceptor of System II becomes accessible to the added hydrophilic reagents even in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea.     

Unsymmetrical 1,1′-disubstituted Ferrocenes: Synthesis of Co(ii), Cu(ii), Ni(ii) and Zn(ii) Chelates of Ferrocenyl -1-thiadiazolo-1′-tetrazole, -1-thiadiazolo-1′-triazole and -1-tetrazolo-1′-triazole with Antimicrobial Properties

Condensation reactions of 1,1"-diacetylferrocene with different heteroaromatic amines such as, 2-amino-1,3,4-thiadiazole, 5-aminotetrazole and 3-amino-1,2,4-triazole to form unsymmetrically 1,1′-disubstituted ferrocenes have been studied. The obtained compounds have been further investigated for their liganding and biological properties upon chelation with Co(II), Cu(II), Ni(II) and Zn(II) metal ions. The synthesized compounds have been characterized by physical, spectral and analytical data and have been screened against pathogenic bacterial strains e.g., Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, showing moderate activity as antibacterials in vitro.     

The rate of polymerase release upon filling the gap between Okazaki fragments is inadequate to support cycling during lagging strand synthesis

Upon completion of synthesis of an Okazaki fragment, the lagging strand replicase must recycle to the next primer at the replication fork in under 0.1 s to sustain the physiological rate of DNA synthesis. We tested the collision model that posits that cycling is triggered by the polymerase encountering the 5′-end of the preceding Okazaki fragment. Probing with surface plasmon resonance, DNA polymerase III holoenzyme initiation complexes were formed on an immobilized gapped template. Initiation complexes exhibit a half-life of dissociation of approximately 15 min. Reduction in gap size to 1 nt increased the rate of dissociation 2.5-fold, and complete filling of the gap increased the off-rate an additional 3-fold (t_1/2  ∼ 2 min). An exogenous primed template and ATP accelerated dissociation an additional 4-fold in a reaction that required complete filling of the gap. Neither a 5′-triphosphate nor a 5′-RNA terminated oligonucleotide downstream of the polymerase accelerated dissociation further. Thus, the rate of polymerase release upon gap completion and collision with a downstream Okazaki fragment is 1000-fold too slow to support an adequate rate of cycling and likely provides a backup mechanism to enable polymerase release when the other cycling signals are absent. Kinetic measurements indicate that addition of the last nucleotide to fill the gap is not the rate-limiting step for polymerase release and cycling. Modest (approximately 7 nt) strand displacement is observed after the gap between model Okazaki fragments is filled. To determine the identity of the protein that senses gap filling to modulate affinity of the replicase for the template, we performed photo-cross-linking experiments with highly reactive and non-chemoselective diazirines. Only the α subunit cross-linked, indicating that it serves as the sensor.     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.