Phytotoxicity of Phthalate Plasticisers: 1. DIAGNOSIS AND COMMERCIAL IMPLICATIONS

The toxicity caused by a volatile constituent from certain samplesof flexible polyvinyl chloride (PVC) was due to dibutyl or diisobutylphthalate (DBP or DIBP) plasticisers. It has caused seriousfinancial losses in the horticultural industry. The two phthalateesters have low volatilities, so any toxicity lasts for manyyears. Radish (Raphanus sativus L. cv. Cherry Belle) seedlings,exposed to an air stream containing 160–180 ng dm^–3of butyl phthalates developed chlorotic leaves within 3–4d and died within 12 d. Neither dioctyl nor diisodecyl phthalate(DOP nor DIDP) produced damage in the test plants. Measurementsof photosynthetic and respiratory gas exchange in intact shootsof affected radishes showed that photosynthesis was severelyinhibited whilst respiration was virtually unaffected. Electronmicrographs of sections from young leaves showed disruptionof thylakoid formation and granai stacking. In mature leaves,thylakoids and grana were well formed but chloroplasts wereswollen and the thylakoids were pushed towards the vacuolarside of the chloroplast. Sensitivity to toxic phthalates variesbetween species; all members of the Cruciferae tested were susceptible,tomato less so, and lettuce and ryegrass were resistant. Toxicityof DIBP, from PVC glazing strip, caused a reduction in cropvalue of ?20 000 per acre per year in commercially grown, monocroptomatoes. Key words: Phthalates, plasticiscd PVC, radish bioassay, glasshouse, tomato, toxicity     

INSECTICIDES: THEIR ROUTE OF ENTRY, MECHANISM OF TRANSPORT AND MODE OF ACTION

(1) The assumption that the circulatory system of the insect is instrumental in transporting insecticides to their site of action appeared not to be based on good evidence. On the contrary, experiments specifically designed to test the hypothesis provided ample evidence to refute the idea. Salient points to disprove the haemolymph route of entry can be summed up as follows: (a) A topically applied dose of insecticide does not readily penetrate the insect and the minor fraction that does so is largely retained in the body wall. The small amount that actually passes into the blood is too small to cause symptoms of toxicity if injected into the haemocoel. (b) The amount of insecticide present in the haemolymph (and CNS) does not appear to have any bearing on toxicity - internally introduced insecticides are, in fact, basically very much less toxic than those externally applied. Where injected doses appear to be more toxic than equal amounts topically applied, this is due to the boosting effect of organic solvent carriers. Tests with parabiotically joined insects provide support for the view that haemolymph-borne insecticide is of no consequence. (c) The topographical proximity of the locus of external application with the site of action (the thoracic ganglia) seems to be important irrespective of the general direction of the blood flow, and this should not be so if the circulatory system was instrumental in the transport of insecticide. (d) The introduction into the haemocoel of material such as olive oil, which is an excellent absorbent for insecticides, does not affect the toxicity (speed of action) of externally applied compounds to a significant extent. It should have a pronounced effect if haemolymph-borne insecticide were an essential element in the process of poisoning. (e) As judged by speed of action, blocking of the blood circulation does not hamper the insecticide's movement to the site of action. (2) Only two other feasible routes remain. (a) The insecticide might reach the CNS via peripheral nerves and nerve cord, but the results of histochemical assays of cholinesterase inhibition in the insect's CNS make the idea improbable for organo-phosphates. The nerve route is also incompatible with the observation that a wax barrier blocked the movement in and over the body wall so as to delay the onset of symptoms of toxicity, as such a barrier would not hinder movement into lateral nerves near the locus of application. (6) The only other feasible alternative, i.e. entry by means of lateral transport via the integument of the body wall and tracheae, is supported by autoradiographic and other evidence which showed the insecticide to accumulate in the tracheal system. It is further supported by the fact that inter-tracheal introduction is faster acting than topical treatment, indicating that the tracheal system offers a very effective pathway to the internal organs. (3) Regarding the mechanism of entry, earlier reviews and text books maintain this to be associated exclusively with penetration into and through the integument by a physicochemical process. However, there is good evidence to show that an active process (requiring metabolic activity as the driving force) plays an essential part in lateral movement in the integument - the epidermis, being the only living tissue continuous throughout the general integument, must perform this function. (4) As to the mode of action, the new hypothesis expounded here implies a single mode based on the fact that insecticides cause the extrusion of fluid from the epidermis into the cuticle and beyond, fluid lost from the epidermal cell layer being replaced from haemolymph and internal tissues. The precise mechanism is not clear but could conceivably involve an as yet hypothetical local endocrine system designed to keep the water content in the integument within certain limits. It is suggested that water extrusion affects the integument's permeability to respiratory gases, resulting in a rate of respiration not commensurate with metabolic need, and that the insecticide's arrival in the trachea (tracheoles) of the CNS leads to excitation and knockdown. Death is thought most likely to be due to dehydration of the CNS and subsequent histological degeneration.     

THE CELLULAR SITE OF ACTION OF ANGIOTENSIN

The site of action and the distribution of angiotensin II have been studied in the mouse. A comparison of the ratios of angiotensin-¹⁴C and inulin-³H at the time of the pressor effect reveals an extracellular pattern of distribution. Morphological studies were made using angiotensin coupled to exogenous enzymes which can be demonstrated histochemically. Coupling of angiotensin to horseradish peroxidase or cytochrome c, with glutaraldehyde or difluorodinitrodiphenylsulfone (FNPS) as the coupling agent, does not alter the pattern of its vasopressor response or that of its inactivation; nor are differences present between angiotensin and the angiotensin-enzyme complexes in the stimulation of in vitro tissue preparations. Dissociation of the complexes was shown not to occur in vitro, but the possibility of a serum factor splitting the complexes immediately after intravenous injection cannot be excluded. Since these complexes are localized on the endothelium and not on the smooth muscle at the time of maximum hypertension, the endothelium is proposed as the site of action for angiotensin.     

THE MODE OF ACTION OF VITAMIN D

1. The purpose of this review article is to re-evaluate and integrate many of the observations related to the physiological effects of vitamin D, using as a working hypothesis the concept that the vitamin may be acting analogously to a steroid hormone in terms of its ability to interact with genetic information and ultimately elicit a physiological response. Prior to this time the problem of the mechanism of action of vitamin D has primarily been approached from the point of view that the vitamin was acting as a cofactor for some specific enzymic reaction. 2. The physiological activities of vitamin D are integrated with those of parathyroid hormone to provide a homeostatic control for the regulation of primarily calcium and secondarily phosphate metabolism. It is proposed that the role of vitamin D in this homeostatic control mechanism is older and more fundamental than that of parathyroid hormone. The interaction of vitamin D on skeletal calcium metabolism may have evolved before the effects of the vitamin on intestinal calcium absorption. 3. There are several physiological defects of calcium metabolism—rickets, osteo-malacia, vitamin D-resistant rickets and idiopathic hypercalcaemia—all of which may be a consequence of an aberration in one or another of the interlocking steps of the vitamin D-dependent and calcium-dependent homeostatic control mechanism. 4. The most thoroughly established action of vitamin D in vivo is to promote or facilitate the intestinal absorption of calcium. Although the exact biochemical details of this process are not available, this may involve vitamin D-mediated synthesis of the appropriate enzyme systems or the alteration of membrane structure necessary for calcium absorption. It is not yet unequivocally established whether calcium absorption is an energy-dependent active transport process or is a passive carrier-mediated or simple diffusion process. 5. The exact action of vitamin D on bone metabolism is not as well established, but the primary effect of the vitamin is likely to mediate bone resorption. The vitamin D-dependent activities of the cell in both the intestine and bone are to absorb calcium and transfer it to the blood. 6. No direct effects of vitamin D on intestinal absorption of phosphate have been found. Furthermore the validity of a vitamin D-mediated renal reabsorption of phosphate is questioned, for the major effects of vitamin D are cation oriented. If the renal effects of vitamin D are true, it is postulated that the mechanism of action of the vitamin here on the anion, phosphate, is fundamentally different from its cation oriented mechanism. 7. There is a lag in the action of vitamin D on the vitamin mediated: (a) transport of calcium both in vivo in rats and chicks, and in vitro with everted intestinal slices; (b) the apparent increased permeability of intestinal mucosa; (c) increased levels of citric acid in serum or bone; (d) the increased incorporation of radioactive inorganic phosphorus into intestinal mucosa phospholipids. As shown by the use of radioactive vitamin D, this lag is not due to a lack of the vitamin in the target organs. 8. Whereas large, unphysiological doses of radioactive vitamin D localize in all tissues and all subcellular fractions, small physiological doses of radioactive vitamin D localize predominantly in the nucleus of the intestinal mucosa. The amount of vitamin D localized in the nucleus would appear to be too low for the vitamin to function as a cofactor, and is more indicative of an interaction on or with deoxy-ribonucleic acid. 9. Actinomycin D, an inhibitor of DNA-directed RNA synthesis, inhibits the action of vitamin D in mediating intestinal calcium absorption and bone resorption. Vitamin D also stimulates messenger-RNA synthesis in intestinal mucosa within 1/2 hr. of vitamin treatment. Vitamin D may play a crucial role, along with parathyroid hormone and calcium, in a DNA, gene-dependent, homeostatic control mechanism for cal, ium metabolism. In this system the vitamin D molecule has certain very specific structural requirements which are probably a reflection of the specificity of its receptor molecule, rather than structural requirements for a cofactor-enzyme relationship.     

动脉壁脑啡肽的含量,存在部位及作用途径

用放射免疫法测得兔动脉的亮氨酸脑啡肽(L-ENK)和甲硫氨酸脑啡肽(M-ENK)样免疫活性物的含量(pg/mg组织湿重)分别为耳动脉38.99±17.29,134.67±8.11;肾动脉31.10±7.76,93.60±18.22,肠系膜动脉25.70 13.60,88.43±18.16。动物经注射交感神经末梢化学切割剂6-OHDA后,这三种动脉中L-ENK样免疫活性物的含量均明显下降(P<0.001);切除颈上神经节使支配耳动脉的交感神经溃变后,或离体耳动脉条受电场刺激后,脑啡肽(ENK)样免疫活性物的含量也都显著下降(P<0.001);但注射利血平则无明显影响,用荧光法测定培育动脉条的浴槽液中NE的含量,观察到ENK可抑制电场刺激所引起的NE的释放。结果提示,动脉壁的L-ENK和M-ENK存在于交感神经末梢中,它可因受电场刺激而释放,可能通过激活突触前膜阿片受体,减少交感神经末梢释放NE,从而抑制动脉的收缩活动。     

大鼠,兔肺动脉壁亮氨酸脑啡肽的分布,含量及其作用途径

用ＡＢＣ免疫组化法对ＳＤ大鼠肺动脉全层漂染,结果显示有亮氨酸脑啡肽（Ｌ－ＥＮＫ）阳性纤维走行于动脉壁外膜中。放免法测得大鼠、兔肺动脉壁合Ｌ－ＥＮＫ,含量（ｐｇ／ｍｇ组织湿重）分别为４３９．１８±３０．５２,２９．９±１．４。离体灌流的兔肺动脉螺旋条,经参数Ⅰ电场刺激可引起收缩反应,α受体阻断剂酚妥拉明可阻断之;α２受体阻断剂育亨宾小剂量增强收缩反应,大剂量则抑制,阿片受体阻断剂纳洛酮可使收缩反应增强。应用酚妥拉明后,参数Ⅱ电场仍能引起较高的收缩反应,纳洛酮对此收缩无影响。外源性ＮＥ引起肺动脉条收缩,Ｌ－ＥＮＫ对之没有抑制效应。结果提示,肺动脉壁含有内源性Ｌ－ＥＮＫ,可因电场刺激而释放,由阿片受体介导抑制动脉壁神经末梢ＮＥ释放,进而抑制血管收缩,对血管平滑肌本身活动及已释放ＮＥ均无抑制效应。关键词：     

Dormancy in Seeds of Charlock: III. OCCURRENCE AND MODE OF ACTION OF AN INHIBITOR ASSOCIATED WITH DORMANCY

The dormancy of charlock seeds appears to be associated withthe presence of an inhibitor which accumulates within the seed.This inhibitor diffuses into the external solution when seedsare placed in water under germination conditions or controlledexperimental conditions. A small quantity of inhibitor diffusesfrom the seed coats but most arises from the embryo. The increasein temperature was measured by comparing the relative growth-ratesof the radicles of excised charlock embryos in water and intest solutions of diffusate. The results suggest that the rateof accumulation of inhibitor in the external solution is controlledby diffusion, and that there is continuous production of inhibitorin the tissues of the embryo which have a low oxgyen tension.The critical concentration of the inhibitor which completelyprevents cell elongation is rapidly attained in seeds whichare dormant. The inhibitor is unlikely to be a mustard oil,such as allylisothio-cyanate.     

STUDIES IN THE MODE OF ACTION OF INSECTICIDES

Homogenates of the thoracic nervous system of Locusta migratoria migratoriodes are able to hydrolyse acetylcholine (ACh) and o -nitrophenylacetate (NPA), and this hydrolysis can be inhibited by tetraethylpyrophosphate (TEPP) at approximately the same molar concentration for both substrates. It is possible that one acetyl-esterase is responsible for the breakdown of the two substances, and there is no reason to assume the existence of a specific acetylcholinesterase. In normal horse serum, on the other hand, the pseudocholinesterase is quite distinct from the enzyme responsible for the breakdown of NPA.
In an attempt to correlate the inhibition of the locust nerve cord acetylesterase with toxic activity to insects and mice, four chlorinated diethyl-phenylphosphates were tested as contact poisons against a number of insects and by injection against locusts and mice, and also as in vitro inhibitors of locust nerve cord acetylesterase and horse-serum pseudocholinesterase. The chemicals were the 2-chloro-, 4-chloro, 2:4-dichloro- and 2:4:5-trichloro- analogues of diethyl-phenylphosphate.
Good correlation exists between their in vitro activity against the nerve-cord acetylesterase and their contact activity to aphids, but not between the former and injection toxicity to locusts. No correlation could be established between the inhibition of horse-serum cholinesterase and injection toxicity to mice. It is thought likely that the inhibition of nerve-cord acetylesterase is of greater importance in aphids than in other insects, where the toxic action of the phosphoric esters is at least partly concerned with other vital processes, and that a detoxication mechanism in the mammal breaks down some of the phosphoric esters, but not others.     

STUDIES IN THE MODE OF ACTION OF INSECTICIDES

Acetylcholine, choline chloride, acetyl-β-methylcholine, benzoylcholine, carbamyl choline, adrenaline and d -tubocurarine are non-toxic when injected into the locust. Prostigmine is also non-toxic, and eserine considerably less toxic to the locust than to man.
The toxic effect of tetraethyl pyrophosphate (TEPP) cannot be antagonized by injection of atropine or enhanced by d -tubocurarine.
The injection of acetylcholine chloride following injection of TEPP does not affect subsequent mortality.
These findings are discussed, and it is suggested that the physiology of the nervous system of the insect is unlike that of the mammal, neither cholinesters nor adrenaline being concerned in it. Phosphorus insecticides are thought to inhibit a general esterase not specifically connected with cholinesters.     

EVIDENCE ON THE SITE OF ACTION OF GROWTH RETARDANTS

1. The ability of five growth retardants to inhibit the GA-inducedand endogenous growth of Avena leaf sections has been investigated.The retardants vary in effectiveness. The order, from most effectiveto least, is Phosfon D, Amo-1618, C011, CCC and B995. 2. The inhibition of growth caused by Phosfon D and Amo-1618is not reversed by GA. It is apparent that the retardants donot compete with GA at the site of GA-action. 3. Addition of IAA will partially reverse the inhibition inducedby Phosfon D or Amo-1618. It is concluded that the retardantsact in part in Avena leaf sections by interfering with the auxinmetabolism of the tisssue. ¹ Supported in part by grants G-14578 and GB-1950 from the NationalScience Foundation ² Present address: Department of Botany, University of Washington,Seattle, Washington     

MODE OF ACTION OF MALEIC HYDRAZIDE ON THE GROWTH OF ESCHERICHIA COLI AND AVENA COLEOPTILE SECTIONS

1. MH was found to suppress the growth and respiration of E. colias well as the IAA-induced growth of Avena coleoptile sections.
2. These suppressions could be reversed more or less strikinglyby the addition of a trace of heavy metals such as Co, Mn, Ni,Zn, Cu, or Mo.
3. The reversal could also be achieved by cysteine,thioglycollate,or fumarate, the latter two substances being,however, lesseffective.
4. The inhibition of the growth of E.coli by MH was completelyrelieved by the addition of IAA. Conversely,the inhibitionof the microbial growth by high concentrationsof IAA couldbe relieved by the addition of MH.
5. It was inferredthat MH may block certain heavy metal-catalyzedprocess, inwhich some thiol substance and IAA are participating,probablyby combining with the heavy metal.

(Received June 23, 1960; )     

精胺抑制人精子的体外受精能力

以精子穿透去透明带仓鼠卵试验(SPA)为模型,评价了精胺对人精子体外受精能力的影响。精胺(0.25—8.0mmol/L)可抑制人精子体外获能和受精,其抑制作用与精胺浓度呈正相关,此种抑制作用是可逆的。用 HPLC 测定精子精胺含量表明,精子获能后精胺含量明显下降。dbcAMP(0.5—1.0mmol/L)或咖啡因(10mmol/L)可拮抗精胺抑制人精子体外获能。其拮抗作用随 dbcAMP 浓度而增强。钙离子载体 A 23187 2/μmol/L 或胰蛋白酶0.05%均可拮抗精胺抑制人精子穿卵率。上述结果提示,精胺可能通过降低精子 cAMP 含量和抑制钙内流或顶体酶活性,从而阻止人精子体外获能和受精。     

Studies in the Physiological Action of 2,2-Dichloropropionic Acid: III. FACTORS AFFECTING THE LEVEL OF ACCUMULATION AND MODE OF ACTION

The factors determining the pattern of uptake by Lemna minorof 2,2-dichloropropionic acid (DCPA), containing chlorine-36,have been examined. Entry takes place via both the roots andfrond and is largely in the ionic form. Initially, the net ratesare high and are replaced by slower but steady rates. It isconcluded that over the first 30 minutes, net uptake is dependenton the physico-chemical processes since (i) the rate is directlyproportional to the external concentration, (ii) on transferenceto buffer containing non-radioactive DCPA up to 90 per centof the radioactivity is exchangeable, (iii) the temperaturecoefficients for the rates of net influx and efflux range from1.2 to 1.4. During the second phase, the net rates of uptakeare curvilinearly related to concentration, the temperaturequotients are higher and the rates of efflux markedly lowerthan in the first phase. Phenylmercuric nitrate and cupric sulphateat 5x10^–6 M halve the net rate of uptake but higher concentrationsare demanded for dinitrophenol, arsenite, aside, and iodoacetate.The depression induced by phenylmercuric nitrate can to somedegree be reversed by the addition of either glutathione orcysteine. For pyruvic acid to halve the rate of uptake of DCPA,it requires more than a three hundredfold greater concentration.It is postulated that in the second phase, uptake is mediatedby metabolic processes involving thiol groups. The ability ofcalcium pantothenate and ß-alanine to reverse theinhibitory effects of DCPA on the growth of L. minor were investigatedin multifactorial experiments. Over a narrow range of concentrations,calcium pantothenate partially offset the inhibitions of growthbut the interactions for ß-alanine were not significant.These results do not support the view that the primary actionof DCPA is to interfere with the biosynthesis of Co-enzyme A.In a comparison of the maximal capacity of different speciesto accumulate DCPA in their roots, up to a six-fold differencewas recorded in the eight species examined. On the basis ofthese findings, the mechanisms determining selective actionand the inhibition of meristematic activity are discussed.