Concurrent evaluation of personal damaging and beneficial UV exposures over an extended period.

Personal exposure to solar ultraviolet radiation (UVR) is acknowledged as having both positive and negative effects on human health. This study aimed to measure concurrently the personal erythemal UV, UVA and vitamin D effective exposures of participants in each season of a year. Participants were all indoor office workers located at two different sites less than 6.5 km apart at the sub-tropical location of Toowoomba (27°33'S 151°55'E). The subjects wore a combined dosimeter badge horizontally on the shoulder for a minimum of one week in each season; this badge used 8-methoxypsoralen film to record the UVA waveband and polyphenylene oxide film for the erythemal and the vitamin D effective UV wavebands. The results show that median erythemal exposure was highest during the spring and lowest during winter, as was the vitamin D effective exposure. Median UVA exposures were at a similar level in winter and summer, autumn was higher (double) and spring at a lower level. The duration and time of day participants spent outdoors changed in each season; in winter, participants spent an average of 101 minutes outdoors between 10:00-14:00 h over the week, whereas in summer this fell to 79 minutes even though they were outdoors more often. The daily UVA/UVB ratio is lowest between 10:00-14:00 h and also changes with the season resulting in the differences between the distributions of exposure for each of the wavebands. Each category of exposures must be assessed individually as each season and each waveband has different distributions. The results also demonstrate that the dual film dosimeter developed and characterized with a calibration to three different biological responses, is an effective device for the concurrent measurement of erythemal UV, UVA and vitamin D effective UV exposures for periods of up to seven days.

A phase-dependent delay of the chick pineal rhythm in rate of thymidine incorporation by brief exposure to aphidicolin.

Cultured chick pineal glands show a persistent rhythm in the rate of cumulative incorporation of thymidine into DNA. In this study we have examined the effects of pulse-exposure to aphidicolin in the dark on the first day of culture on thymidine incorporation during the second and third days of culture in the dark with aphidicolin-free medium. The phase of the rhythm in the rate of thymidine incorporation was delayed in a concentration-dependent manner by a 4-hr exposure to aphidicolin, commencing in the final hour of the photoperiod. This effect was phase-dependent and not seen when exposure to aphidicolin began earlier in the photoperiod.

A phase-dependent delay of the chick pineal rhythm in rate of thymidine incorporation by brief exposure to ouabain.

Cultured chick pineal glands show a persistent rhythm in the rate of cumulative incorporation of thymidine into DNA [Wainwright and Wainwright, 1989]. In this study we have examined the effects of a pulse-exposure to ouabain in the dark on the first day of culture upon thymidine incorporation during the second and third days of culture in the dark with ouabain-free medium. The phase of the rhythm in rate of thymidine incorporation was delayed by a 4-hr exposure to 100 microM ouabain commencing in the final hour of the photoperiod, but not by a 2-hr exposure or by 10 microM ouabain. This effect was phase-dependent and not seen when exposure to ouabain began earlier in the photoperiod. The phase delay caused by ouabain was not due to a persistent direct inhibition of the process of thymidine incorporation. The phase-shift due to exposure to ouabain was superimposed on a phase delay caused by renewal of culture medium 4 hr after explanting the glands into culture.

Inhibition of the chick pineal rhythm in rate of thymidine incorporation by cyclic AMP.

Chick pineal glands in organ culture showed a circadian rhythm in the rate of thymidine incorporation. Thymidine incorporation was very markedly inhibited when 3-isobutyl-l-methylxanthine (IBMX) was continuously present. When IBMX was added to cultures in control medium during the photoperiod of the second day in culture, the extent of inhibition of incorporation during that photoperiod increased with the increase in length of the photoperiod remaining. Incorporation did not resume at the start of a second photoperiod if IBMX was added within the first 10 h of the first photoperiod. Corresponding results were obtained with glands continuously cultured in constant darkness. Similar results were also obtained using glands treated with 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), 7 beta-acetoxy-8,13-epoxy-1 alpha, 6 beta, 9 alpha-trihydroxy-1abd-14-ene-11-one (forskolin), or 8-bromo-cyclic AMP, but not with 8-bromo-cyclic GMP. When glands cultured with IBMX were transferred to control medium, incorporation remained inhibited until the start of the next photoperiod. We conclude that the increase in the rate of thymidine incorporation at the start of each new photoperiod is dependent on a "switch" process that is inhibited by elevated concentrations of cyclic AMP.

Thymidine kinase activity of the chick pineal gland.

Pineal thymidine kinase activity of 1-week-old chicks in situ varied significantly throughout the day. However, the circadian rhythm of thymidine incorporation seen with cultured chick pineal glands was not accompanied by variations in level of thymidine kinase activity in vitro. Thus the circadian rhythm in rate of cumulative incorporation of thymidine by cultured chick pineal glands is not determined by a rhythm in rate of the first reaction of the complex series of reactions by which thymidine is incorporated into DNA.

Purinergic receptors have no major role in control of the circadian rhythm in rate of thymidine incorporation by cultured chick pineal glands.

We have examined the effects of some analogues of adenosine upon the circadian rhythm in rate of thymidine incorporation by cultured chick pineal glands. Incorporation in the early period of the photoperiod on day 2 of culture was slightly inhibited by the adenosine analogue N-ethylcarboxamido-adenosine, but this effect was not countered by the antagonist 8-phenyl-theophylline. Thymidine incorporation was inhibited when glands were continuously exposed to the adenosine transport inhibitor nitrobenzyl-thioinosine, but ongoing incorporation was not inhibited by addition of this agent. Removal of adenosine and deoxyadenosine supplements from the medium, with or without further addition of adenosine deaminase, had no appreciable effects upon thymidine incorporation. We conclude that adenosine and analogues probably play no role in regulation of the rhythm in rate of thymidine incorporation.

Thymidine incorporation by chick pineal glands as studied by pulse-labelling in vitro.

We have studied the pattern of variations in extent of thymidine incorporation during pulse-labelling in cultured chick pineal glands. During the first 24-30 h in culture the extent of pulse-labelling varied in parallel with the cycle in cumulative incorporation. Pulse-labelling was seen during the period of apparent arrest of cumulative incorporation. However, it was probably "masked" by the SD of assays of cumulative incorporation and represented a minor, but distinct, process of thymidine incorporation. The pattern of variation in extent of pulse-labelling was qualitatively consistent under a wide variety of conditions. Control of this pattern appeared to be different from that of the circadian rhythm in cumulative thymidine incorporation.

Entrainment of the rhythm in thymidine incorporation by cultured chick pineal glands.

Young chicks were raised under a standard cycle of illumination and their pineal glands were cultured in organ culture. We have already reported a rhythmic daily cycle in the cumulative incorporation of thymidine into DNA when the glands were incubated under the cycle of illumination to which they were entrained in vivo. Incorporation ceased shortly after the end of the photoperiod and resumed again at, or shortly after, start of the following photoperiod. We have now shown that this cycle was entrained to other lighting schedules and largely unaffected by the time of explanting into culture. The rhythm also persisted in glands cultured in constant darkness. However, when the cycle was maintained by daily renewal of the culture medium it was markedly affected by changes in the time of day at which medium was renewed. Results for glands cultured under constant illumination were inconsistent.

Thymidine incorporation by cultured chick pineal glands is not subject to adrenergic regulation.

Norepinephrine is known to play a role in regulating the circadian rhythms of serotonin N-acetyltransferase activity and melatonin formation in the chick pineal gland. We have recently demonstrated that the cultured chick pineal exhibits a circadian rhythm in the incorporation of thymidine. In this study we show that this latter rhythm is not subject to adrenergic control.

Rhythmic incorporation of thymidine by chick pineal glands in vitro.

Cultured chick pineal glands showed a cycle in the cumulative incorporation of thymidine into DNA. In undisturbed cultures the rate of thymidine incorporation, amount of thymidine incorporated per 1-day cycle, and persistence of the incorporation process were all markedly affected by the concentration of exogenous precursor. However, more than two full cycles of incorporation were found when culture medium of low thymidine content was renewed daily, or when medium of intermediate concentration was replaced on the 3rd day of culture. At a high thymidine concentration the second cycle of incorporation sometimes appeared to be impaired. At all concentrations tested, less than 2% of the available thymidine was incorporated.

On the uptake and incorporation of thymidine by cultured chick pineal glands.

Cultured chick pineal glands showed a marked cycle in the incorporation of thymidine into DNA. They also accumulated exogenous thymidine in one, or more, endogenous thymidine pool(s). However, there was no significant variation in ability to take up thymidine during incubation with either standard medium or media of increased thymidine content. We could not eliminate the possibility of variation in size of a quantitatively minor, but metabolically extremely important, thymidine pool which "channels" precursor directly into DNA.

On thymidine incorporation by the cultured chick pineal gland.

Explanted chick pineal glands exhibited a cycle in thymidine incorporation when cultured either under a cycle of illumination or in constant darkness. This cycle appeared to be entrained to the light cycle under which the birds were maintained. The incorporation reflected gene replication in a small fraction of the cell population that was largely, but not entirely, located in the stroma of the gland. Glands cultured with colchicine for 28 h contained a very small number of cells showing metaphase chromosomes.

Effects of some inhibitors of DNA synthesis and repair upon the cycle of serotonin N-acetyltransferase activity in cultured chick pineal glands.

When chick pineal glands were cultured in the dark with aphidicolin from midphotoperiod, the increase of serotonin N-acetyltransferase (NAT) activity was stimulated and the time of peak NAT activity was advanced. The peak level of NAT activity was also reached sooner on the 2nd day of culture. The increase of NAT activity was also stimulated in glands cultured under diurnal illumination, but the time of peak activity was not advanced. Effects with glands explanted into culture in the dark at other times were smaller and the time of peak NAT activity was not changed. Cytosine arabinoside and dideoxythymidine also stimulated the increase of NAT activity and advanced the time of peak activity with glands cultured in the dark from midphotoperiod. 3-Aminobenzamide markedly stimulated the increase of NAT activity both in the dark and under diurnal lighting when pineal glands were explanted into culture at mid- or late photoperiod. In contrast, with glands in culture from earlier in the photoperiod, aminobenzamide had no effect upon the increase of NAT activity up to the peak level found with control glands. Thereafter results were variable. Effects of cordycepin upon development of NAT activity were similar to those of 3-aminobenzamide but less marked. Incorporation of thymidine into acid-insoluble material in the dark was very markedly inhibited by aphidicolin, cytosine arabinoside, and dideoxythymidine, but only slightly by cordycepin. Aminobenzamide strongly inhibited incorporation by glands cultured from midphotoperiod, but had little effect with glands in culture from near the end of the photoperiod. We adopt the working hypothesis that excision repair of DNA may be a major component in the mechanism of the chick pineal clock.

Interdependent effects of the ionophore A23187 and serum on the serotonin N-acetyltransferase activity of cultured chick pineal glands.

Marked effects of the ionophore A23187 on the cycle of N-acetyltransferase (NAT) activity in cultured chick pineal glands were observed under three conditions of illumination. However, the effects were qualitatively and quantitatively dependent on the batch of fetal calf serum used in the medium and time of explanation into culture. Ionophore increased the level of NAT activity remaining in glands exposed prematurely to light regardless of the serum used. The ionophore suppressed the "spike" in cyclic GMP content of glands cultured in the dark, and extended the period of maximum cyclic GMP content of glands under diurnal illumination.

Effects of some fatty acids on the serotonin N-acetyltransferase activity in cultured chick pineal glands.

Low concentrations of arachidonate, oleate, or palmitate significantly affected the cycle of NAT activity of cultured chick pineal glands in different ways. The effects observed were altered by change of the lighting conditions of culture. Effects of arachidonate were also shown to be altered by change of the serum component of the culture medium. Effects of premature exposure to light of glands cultured under diurnal conditions of illumination were changed markedly by substitution of the serum component of the medium or a supplement of ionophore A23187 and less markedly by supplements of fatty acids.

Relationship between cycles in level of serotonin N-acetyltransferase activity and cyclic GMP content of cultured chick pineal glands.

We examined effects of supplements on cycles in cyclic GMP content and serotonin N-acetyltransferase (NAT) activity in cultured chick pineal glands. Increases in cyclic GMP content and NAT activity were stimulated by 1-ethyl-4(isopropylidene-hydrazino)-1H-pyrazolo[3,4-b]pyridene-5-c arboxylic acid, ethyl ester, hydrochloride and isobutylmethyl xanthine under diurnal illumination and in constant darkness, but subsequent decreases were not inhibited. Hypoxanthine had little effect on NAT activity under all lighting conditions, or on the content of cyclic GMP in glands cultured in the dark. However, it markedly stimulated accumulation of cyclic GMP in illuminated cultures. EGTA or additional Ca2+ had no effect on pineal NAT activity. However, EGTA markedly stimulated accumulation of cyclic GMP both in the light and in the dark. Supplementary Ca2+ slightly retarded accumulation of cyclic GMP in the dark but stimulated slightly in the light.

Phase-shifting of cycles in level of serotonin N-acetyltransferase activity and cyclic GMP content of cultured chick pineal glands by methotrexate.

Methotrexate at 1 microM stimulated increase of serotonin N-acetyltransferase (NAT) activity in chick pineal glands cultured under each of three conditions of illumination. The peak of the circadian rhythm in NAT activity and the "spike" in content of cyclic GMP were both advanced in pineal glands cultured in the dark from midphotoperiod. In contrast, the time of peak NAT activity in glands cultured in the dark from late photoperiod was unaffected. In addition, methotrexate did not affect times of reaching maximum NAT activities in glands cultured from midphotoperiod in the light or under diurnal illumination. Doubling the concentration of methotrexate also eliminated the lag phase in increase of NAT activity in glands cultured in the dark. However, at a concentration of 5 microM methotrexate the curve depicting increase of NAT activity was biphasic, and neither time nor level of peak NAT activity differed from those of control glands. Results of attempts to demonstrate persistent effects of exposure to methotrexate were inconclusive.

Neopterin retards loss of serotonin N-acetyltransferase activity from cultured chick pineal glands.

D-Neopterin at 10 microM delayed start of the decline of serotonin N-acetyltransferase (NAT) activity from the peak level in the cycle exhibited by chick pineal glands cultured under standard conditions in the dark. A less marked retardation of decline of NAT activity was found with glands cultured under diurnal illumination or those exposed prematurely to light. There were no significant effects of neopterin on the increases of NAT activity or peak levels of activity developed. The pteridine also retarded loss of NAT activity from the peak level developed in the dark when the time of explanting into culture was later in the (solar) day, but not when it was earlier. Neopterin had no effect on the cycle in cyclic GMP content of cultured chick pineal glands.

Enzymes of guanine nucleotide metabolism and the diurnal cycle in cGMP content of the chick pineal gland.

Levels of cGMP phosphodiesterase, guanylate cyclase, and GTPase activities were determined in homogenates of chick pineal glands. Only small variations in vivo were observed with glands removed at different times of the day from birds under a standard cycle of illumination. Glands cultured under the cycle of illumination from late in the photoperiod showed a progressive loss of about half the phosphodiesterase activity in 24 h, and an increase of roughly 75% in GTPase activity within 12 h. No simple correlations were found between variations in levels of enzyme activity and the diurnal cycles in pineal content of cGMP and level of serotonin N-acetyltransferase (NAT) activity. However, onset of rapid increases in 3',5'-cyclic GMP (cGMP) content and NAT activity was correlated with a transient decrease of about 30% in the phosphodiesterase activity, both in vivo and in culture. Further, known inhibitors of phosphodiesterase activity previously shown to elicit increase of cGMP content and marked elevation of NAT activity in cultured glands only inhibited phosphodiesterase activity of homogenates by 25-30%. It was therefore concluded that the transient decrease in level of phosphodiesterase may facilitate onset of increase in pineal cGMP content. However, it seems improbable that changes in pineal content of enzymes of guanine nucleotide metabolism are essential to regulation of diurnal cycles in cGMP content or level of NAT activity.